Reducing the risk of human anti-human antibodies through V gene manipulation

ABSTRACT

The present embodiments relate to methods of identifying and creating human or humanized antibodies that possess a reduced risk of inducing a Human Anti-Human Antibody (HAHA) response when they are applied to a human host. Other methods are directed to predicting the likelihood of a HAHA response occurring. Methods for screening for anti-HAHA compounds are also included. Methods for determining if various conditions for administering an antibody to a subject enhance or suppress a HAHA response are also included. Some embodiments herein are directed to transgenic mouse embodiments relevant for HAHA responses.

REFERENCE TO RELATED APPLICATION

The present application is a divisional of U.S. application Ser. No.11/136,250, filed May 23, 2005, now U.S. Pat. No. 7,625,549, which is acontinuation in part of U.S. nonprovisional application Ser. No.11/084,554 filed Mar. 17, 2005, now abandoned, and PCT Application No.:PCT/US2005/009306, filed Mar. 17, 2005, both of which claim priority toU.S. provisional application No. 60/554,372, filed Mar. 19, 2004, andU.S. provisional application No. 60/574,661, filed May 24, 2004, all ofwhich are hereby incorporated by reference in their entireties.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledSeqList_ABGENIX-100CPDV.txt, created Sep. 29, 2009, last modified Oct.12, 2009, which is 177,809 bytes in size. The information in theelectronic format of the Sequence Listing is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

Embodiments of the invention relate to the prediction, manipulation, andprevention of immunogenicity and to XenoMouse® animals and other similarorganisms and methods of using them for predicting and altering the riskthat a substance, such as an antibody, or a particular method willinduce a human anti-human antibody response in a patient.

BACKGROUND OF THE INVENTION

The utility of antibodies in the therapy of clinically relevant diseasesis well acknowledged. One of the primary dangers of these antibodies isthe risk of an immune response by the patient, which receives theantibody, e.g., that the patient will make antibodies to the therapeuticantibodies.

Generally, previous research has focused on the risk associated with theaddition of a mouse-based antibody to a human, resulting in the humanpatient launching an immune response against the mouse-based antibody.This immune response has also been termed a HAMA response, for “humananti-murine antibody.”

One attempt to limit this HAMA response is described in U.S. Pub. No.20040005630 to Gary Studnicka (Published Jan. 8, 2004) hereinincorporated in its entirety by reference. This publication disclosespossible methods for how one might compare sequences, on an amino acidlevel, in order to determine which amino acids one might be able tochange without reducing the affinity of the antibody, whilesimultaneously reducing the immunogenicity of the antibody so that onecould administer the altered antibody to heterologous species. Thisreference suggests that the way to overcome the problem of a HAMAresponse is to make a residue by residue comparison of a workingantibody to a consensus sequence. Particular amino acid positions thatare exposed to solvent are then changed, if the residue is not involvedin binding and if that residue is highly or moderately conserved in ahuman consensus sequence.

Others have attempted to determine whether or not V_(H) gene usage iscorrelated with autoimmune diseases in general. These attempts have hadlittle success. One such attempt was made by Huang et al. (Clin. Exp.Immunol. 112:516-527, (1998)). Huang et al. attempted to determine ifthere was some correlation between V_(H) usage in patients withrheumatoid arthritis. Previous studies had resulted in conflictingresults. Huang et al. looked at eight different V_(H)3 genes and threedifferent V_(H)4 genes. However, their conclusion was that usage ofindividual V_(H) genes in peripheral blood B cells was not affected bythe disease. Huang et al. concluded that while there may be some V_(H)genes that are preferentially used in rheumatoid factors, the overallrepresentation of V_(H) genes in the peripheral B cells is not altered.Moreover, these experiments were limited to generalized autoimmuneproblems.

The complexities in attempting to reduce immunogenicity are numerous.For example, several factors to be considered include the following:murine constant regions, V-region sequences, human immunoglobulinallotypes, unusual glycosylation, method of administration, frequency ofadministration, dosage of antibody, patient's disease status, patient'simmune status, patient's MHC haplotype, specificity of antibody, cellsurface or soluble antigen, degree of aggregation of the biologic beingadministered, formation of immune complexes with antigen, complementactivation by antibody, Fc receptor binding by antibody, inflammationand cytokine release. (Mike Clark, Immunology Today, August 2000).However, Clark noted that some of the immunogenicity issues associatedwith V-region sequences can be altered by humanization.

Several studies have addressed polymorphisms and repertoire expressionof V genes, sometimes in relation to ethnicity, age or gender. Thesereports relied on a single or very few donors (Hufnagle et al., Ann N YAcad Sci.; 764:293-295 (1995); Demaison et al., Immunogenetics.,42:342-352 (1995); Wang et al., Clin Immunol., 93:132-142 (1999); Rao etal., Exp Clin Immunogenet., 13:131-138 (1996); Brezinschek et al., JImmunol., 155:190-202 (1995); and Rassenti et al., Ann N Y Acad Sci.,764:463-473 (1995)), analyzed leukemia or autoimmune patients (Dijk-Hardet al., J Autoimmun. 12:57-63 (1999); Logtenberg et al., Int Immunol.,1:362-366 (1989); Dijk-Hard et al., Immunology, 107:136-144 (2002);Johnson et al., J Immunol., 158:235-246 (1997)), focused on a limitednumber of genes (Pramanik et al., Am J Hum Genet., 71:1342-1352 (2002);Rao et al., Exp Clin Immunogenet., 13:131-138 (1996); Huang et al., MolImmunol., 33:553-560 (1996); and Sasso et al., Ann N Y Acad Sci.,764:72-73 (1995)), or categorized V_(H) gene use by family (Hufnagle etal., Ann N Y Acad Sci., 764:293-295 (1995); Rassenti et al., Ann N YAcad Sci., 764:463-473 (1995); and Logtenberg et al., Int Immunol.,1:362-366 (1989); Ebeling et al., Int Immunol., 4:313-320 (1992)).

Unfortunately, even when the HAMA response is eliminated, therapeuticantibodies can still elicit an immune response in patients. In otherwords, the antibody can elicit a human anti-human antibody (HAHA)response. This response can limit the antibodies' efficacy and cannegatively affect their safety profile in the worst-case scenario. As anexample, the fully human phage display-derived anti-TNF antibody HUMIRA®(Abbott Laboratories) unexpectedly provokes a HAHA response inapproximately 12% of patients on monotherapy and about 5% in combinationtherapy with the methotrexate. Thus, while attempts have been made inovercoming the risks associated with a HAMA response, little has beendone to address HAHA response issues.

SUMMARY OF THE INVENTION

Genes that are under-represented in the general population cancontribute to the immunogenicity of a therapeutic antibody having aprotein structure that can be encoded by such a gene. Identification ofthese genes will aid in the assessment of therapeutic candidatemonoclonal antibodies and can be incorporated as a selection factor atthe time of preclinical development. This represents the first study ofa large number of normal donors with respect to the presence and usageof a large number of individual V_(H) and V_(L) genes and how theseindividual genes correlate with the risk of a HAHA response.

One aspect of the invention is a method of selecting an antibody for ahost. The antibody has a decreased likelihood of causing a humananti-human antibody (HAHA) response in the host is provided. The methodcomprises providing an immunoglobulin gene encoding a candidateantibody, providing a host immunoglobulin gene from a host that is toreceive the candidate antibody, comparing the immunoglobulin geneencoding the candidate antibody with the host immunoglobulin gene, andselecting the candidate antibody if the immunoglobulin gene encoding theantibody is the same as the host immunoglobulin gene, thereby selectingan antibody for the host that has a decreased likelihood of causing aHAHA response. In some embodiments, it further comprises repeating thesteps of providing, comparing, and selecting for more than oneimmunoglobulin gene of the candidate antibody. In some embodiments, itfurther comprises repeating the steps of providing, comparing, andselecting for every immunoglobulin V gene of the candidate antibody. Insome embodiments, the immunoglobulin gene is a V gene. In someembodiments, the V gene is a V_(H) (heavy) gene. In some embodiments,the V gene is a V_(L) (light) gene. In some embodiments, providing agene comprises recognizing the identity of the immunoglobulin gene.

Another aspect of the invention is a method of selecting an antibodywith a reduced risk of inducing a human anti-human antibody (HAHA)response for a host. It comprises comparing an antibody V gene set witha host V gene set and selecting the antibody that is encoded by a V geneset that is present in the set of host V genes. In some embodiments, thehost V genes are transcribed in the host. In some embodiments, the hostV genes are translated in the host. In some embodiments, the V genes areV_(H) genes. In some embodiments, the V genes are V_(L) genes.

Another aspect of the invention is a method of excluding an antibodyfrom use in the treatment of a host. The method comprises providing agene encoding at least a part of an antibody, determining if the gene isthe same as a gene in a host to receive the antibody, and excluding theantibody if the gene encoding at least a part of the antibody is notalso a gene in the host. In some embodiments, the method furthercomprises providing all genes encoding the antibody, determining if eachof the genes is the same as any genes in the host, and excluding theantibody if any of the genes is not also a gene in the host. In someembodiments, the antibody is excluded if the gene encoding at least apart of an antibody is a V_(H)3-9, V_(H)3-13, or V_(H)3-64 gene.

Another aspect of the invention is a method of selecting an antibody foradministration to a member of a population, the antibody having areduced likelihood of causing a human anti-human antibody (HAHA)response in the population, comprising providing a V gene encoding atleast a part of a candidate antibody to be administered to an individualin a population, providing a frequency of occurrence for the V gene inthe population, and selecting the candidate antibody if the V gene has ahigh frequency of occurrence in the population. In some embodiments, themethod further comprises providing all of the V genes for the candidateantibody, providing a frequency of occurrence for all of the V genes inthe population, and selecting the candidate antibody if all of the Vgenes have a frequency of occurrence above a predetermined frequency ofoccurrence in the population. In some embodiments, the predeterminedfrequency of occurrence is at least 50% of the population. In someembodiments, the predetermined frequency of occurrence is at least 80%of the population. In some embodiments, the predetermined frequency ofoccurrence is at least 99% of the population. In some embodiments, thepredetermined frequency of occurrence is at least 100% of thepopulation. In some embodiments, the V gene is an immunoglobulin V_(H)type gene or variant thereof. In some embodiments, the V gene is animmunoglobulin V_(L) type gene or variant thereof. In some embodiments,the antibody is from a nonhuman animal that produces human antibodies.

Another aspect of the invention is a method of identifying an antibodywith a high risk of inducing a human anti-human antibody (HAHA) responsein a host. It comprises determining if a gene that encodes the antibodyis one of a V_(H)3-9, a V_(H)3-13, and a V_(H)3-64 gene.

Another aspect of the invention is a method for selecting an antibody.The method comprises determining a frequency with which a gene encodingan antibody occurs in a particular human population and selecting theantibody as a function of the frequency, thereby reducing the risk thatthe antibody will induce a human anti-human antibody response in a humanhost.

Another aspect of the invention is a method of selecting an antibody fora patient in order to reduce the risk that the antibody will induce ahuman anti-human antibody response. The method comprises determining theethnic background of a patient and selecting an antibody comprising aset of V genes that is optimized for the occurrence of a V gene that iscommon in the ethnic background so as to reduce the risk that theantibody will induce a human anti-human antibody response. In someembodiments, the method further comprises a step of initiallydetermining which V genes are common in the ethnic background. In someembodiments, the antibody is selected by comparing substantially all ofthe V genes of the antibody to a normalized host V gene profile for theethnic background.

Another aspect of the invention is a method for determining a risk of ahuman anti-human antibody (HAHA) response occurring for a particularantibody. The method comprises identifying a gene that encodes anantibody, comparing the identity of the gene to a gene profile, andscoring the gene if it occurs with less than a predetermined frequencyof occurrence in the gene profile, wherein a score indicates a risk of aHAHA response occurring for the antibody. In some embodiments, the geneprofile is a gene profile of an individual and the predeterminedfrequency of occurrence is 100%. In some embodiments, the gene profileis a normalized host V gene profile for a population. In someembodiments, the normalized host V gene profile is selected based on anindividual's genetic makeup. In some embodiments, the normalized host Vgene profile is selected based on an individual's ethnic background. Insome embodiments, a V gene in the normalized host V gene profile has afrequency of occurrence that is below the predetermined frequency ofoccurrence and is selected from the group consisting of: V_(H)3-9,V_(H)3-13, and V_(H)3-64. In some embodiments, the predeterminedfrequency of occurrence is selected from the group consisting of: atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 99%, and at least 100%.

Another aspect of the invention is a transgenic animal for identifyingantibodies that have a risk of inducing an immunological response for aparticular human population. The transgenic mouse comprises a transgenicanimal that has been modified to produce human antibodies in response toantigenic challenge, wherein a set of human immunoglobulin genes in thetransgenic animal is the same as a gene set of a particular humanpopulation, and wherein the transgenic animal does not have anyadditional V genes apart from those in the gene set of the particularhuman population. In some embodiments, the transgenic animal has beenmodified to produce fully human antibodies in response to antigenicchallenge. In some embodiments, the set of human immunoglobulin genes inthe transgenic animal is present in at least 50% of the particular humanpopulation. In some embodiments, the set of human genes in thetransgenic animal is present in at least 100% of the particular humanpopulation. In some embodiments, an endogenous loci of the transgenicanimal has been inactivated. In some embodiments, the populationconsists of one person. In some embodiments, the population consistsessentially of a genetically related family. In some embodiments, thepopulation is defined across ethnic groups. In some embodiments, thepopulation is defined within one ethnic group. In some embodiments, thetransgenic animal further comprises an identifier that matches thetransgenic animal with the population. In some embodiments, thetransgenic animal further comprises a fully human or humanized antibody,and the fully human or humanized antibody is foreign to the transgenicanimal.

Another aspect of the invention is a transgenic mouse for use indetecting an antibody with a relatively high-risk of inducing a humananti-human antibody (HAHA) response. It comprises a transgenic mousethat can express a fully human antibody, wherein the transgenic mousecomprises a human immunoglobulin gene set, and wherein the transgenicmouse lacks V genes in the human immunoglobulin gene set that a patientthat is to receive an antibody tested by the transgenic mouse alsolacks, and a human antibody in the transgenic mouse, wherein the humanantibody is to be administered to the patient, and wherein the humanantibody is an exogenous antibody to the transgenic mouse.

Another aspect of the invention is a transgenic mouse for use inidentifying antibodies that will induce an immunological response in aparticular patient population. The mouse comprises a transgenic mousethat is configured to produce humanized antibodies in response toantigenic challenge, wherein the mouse comprises a human immunoglobulingene set, and wherein the human immunoglobulin gene set does not containany high risk genes. In some embodiments, the gene set is a V gene set.In some embodiments, the high-risk genes are V_(H)3-9, V_(H)3-13, andV_(H)3-64. In some embodiments, the V gene set consists essentially oflow-risk genes. In some embodiments, the high risk genes are any genesthat do not occur in 100% of the population. In some embodiments, themouse is used to determine if other substances or variations in methodsincrease the likelihood that a HAHA response will occur. Those thatincrease or decrease the likelihood of a HAHA response can then beidentified. In some embodiments, the variable that is altered is themethod of administration of the antibody, the amount of antibodyadministered, or the number of times that an antibody is administered.In some embodiments, substances to be added include adjuvants, antigenicsubstances, and/or candidate HAHA inhibitors.

Another aspect of the invention is a kit for detecting an antibody thatcan induce a human anti-human antibody (HAHA) response in a patient. Thekit comprises a transgenic mouse that can express a fully humanantibody, wherein the transgenic mouse comprises human immunoglobulingenes, and wherein the transgenic mouse lacks a V gene set that apatient that is to receive an antibody tested by the transgenic mousealso lacks and a means for administering an antibody to the transgenicmouse. In some embodiments, the kit further comprises a means fordetecting a HAHA response in the transgenic mouse. In some embodiments,the kit further comprises an antigenic substance, wherein the antigenicsubstance is associated with an antibody to be tested to see if it willinduce a HAHA response. In some embodiments, the antigenic substance isT cell epitope (TCE).

Another aspect of the invention is a method of selecting an antibody soas to reduce the risk of a human anti-human antibody (HAHA) responsebeing induced in a human. The method comprises administering an antibodyto a transgenic mouse, wherein the transgenic mouse comprises humangenes allowing the mouse to be capable of producing a fully human orhumanized antibody, observing if the antibody results in a HAHA responsein the transgenic mouse, and selecting the antibody if it does notresult in a HAHA response in the mouse. In some embodiments, the methodfurther comprises the step of selecting a different antibody if thefirst administered antibody results in a HAHA response and repeating thesteps until an antibody is observed that does not induce a HAHAresponse. In some embodiments, the antibody is a fully human antibody.In some embodiments, the observing if the antibody results in a HAHAresponse comprises examining a blood sample from the mouse for anantibody that can bind to the administered fully human antibody. In someembodiments, the transgenic mouse comprises the same V genes as thehuman that is to receive the antibody. In some embodiments, the methodfurther comprises the step of first selecting the transgenic mouse basedupon a similarity between a human immunoglobulin gene set in thetransgenic mouse and a human immunoglobulin gene set in the human. Insome embodiments, the transgenic mouse comprises the same V_(L) genes asthe human that receives the antibody. In some embodiments, thetransgenic mouse comprises the same V_(H) genes as the human thatreceives the antibody. In some embodiments, the V genes in thetransgenic mouse consists essentially of the same V genes as the humanthat receives the antibody. In some embodiments, the V_(L) genes in thetransgenic mouse consists essentially of the same V_(L) genes as thehuman that receives the antibody. In some embodiments, the V_(H) genesin the transgenic mouse consists essentially of the same V_(H) genes asthe human that receives the antibody. In some embodiments, thetransgenic mouse is essentially free of high risk genes. In someembodiments, the transgenic mouse essentially consists of low riskgenes. In some embodiments, the transgenic mouse does not have a geneselected from the group consisting of: V_(H)3-9, V_(H)3-13, andV_(H)3-64 genes.

Another aspect of the invention is a method of determining a risk thatan antibody will induce a human anti-human antibody (HAHA) response in apatient. The method comprises administering an antibody to a nonhumananimal that can produce human or humanized antibodies, waiting for aperiod of time sufficient to allow a HAHA response to occur, andobserving if a HAHA response is induced by the antibody. In someembodiments, the nonhuman animal is a transgenic mouse, and wherein allof the somatic and germ cells of the mouse comprise a DNA fragment ofhuman chromosome 14 from the five most proximal V_(H) gene segments,continuing through the D segment genes, the J segment genes and theconstant region genes through C-delta of the human immunoglobulin heavychain locus, wherein the fragment does not contain a C-gamma gene, andwherein the fragment is operably linked to a human C-gamma-2 regiongene. In some embodiments, the method further comprises the step ofselecting a transgenic mouse based on a similarity between the patientimmunoglobulin gene set and the transgenic mouse immunoglobulin geneset. In some embodiments, the similarity is that the gene sets lack asame immunoglobulin gene. In some embodiments, the same immunoglobulingene is a high-risk gene.

Another aspect of the invention is a kit for assessing the risk of ahuman anti-human antibody (HAHA) response being induced by an antibody.The kit comprises a nonhuman animal that comprises a means for producinga fully human antibody, an exogenous antibody to be tested in thenonhuman animal, means for administering the antibody to the nonhumananimal, and means for testing if a HAHA response occurred in thenonhuman animal. In some embodiments, the non-human animal has nohigh-risk genes. In some embodiments, the high-risk genes are selectedfrom the group consisting of: V_(H)3-9, V_(H)3-13, and V_(H)3-64.

Another aspect of the invention is a transgenic mouse for screening foragents that inhibit the induction of a human anti-human antibody (HAHA)response. The mouse comprises a human gene configured to allow thetransgenic mouse to produce a fully human or humanized antibody, and aHAHA inducing antibody in the transgenic mouse. In some embodiments, thetransgenic mouse further comprises a candidate HAHA inhibitor that is inthe transgenic mouse. In some embodiments, the transgenic mouse lacksany high-risk genes. In some embodiments, the high-risk genes areselected from the group consisting of: V_(H)3-9, V_(H)3-13, andV_(H)3-64 and a combination thereof. In some embodiments, the HAHAinducing antibody is encoded by a high-risk gene. In some embodiments,the high risk gene is selected from the group consisting of: V_(H)3-9,V_(H)3-13, V_(H)3-64, and some combination thereof.

Another aspect of the invention is a method for screening for agentsthat inhibit the induction of a human anti-human antibody (HAHA)response. The method comprises administering a HAHA inducing antibody toa transgenic mouse, the transgenic mouse comprising a human geneconfigured to allow the transgenic mouse to produce a fully human orhumanized antibody, administering a candidate HAHA inhibitor to thetransgenic mouse, and observing if a resulting HAHA response isinhibited after an amount of time sufficient to allow for a HAHAresponse. In some embodiments, the HAHA inducing antibody is an antibodyencoded by a high-risk gene. In some embodiments, the HAHA inducingantibody is an antibody with a high-risk V gene. In some embodiments,the HAHA inducing antibody is created by a transgenic mouse capable ofmaking fully human antibodies. In some embodiments, the HAHA response ismonitored through the production of an antibody that binds to the HAHAinducing antibody. In some embodiments, the candidate HAHA inhibitor isadministered to the transgenic mouse before the HAHA inducing antibodyis administered to the transgenic mouse. In some embodiments, more thanone candidate HAHA inhibitor is administered to the transgenic mouse.

Another aspect of the invention is an antibody composition comprising afully human or humanized antibody and a molecule of a T cell epitope(TCE), wherein the molecule of TCE is connected to the antibody. In someembodiments, the antibody composition further comprises more than oneantigenic substance attached to the antibody. In some embodiments, theantigenic substance is attached to the antibody by a maleimide group.

Another aspect of the invention is a method of increasing theprobability that a human anti-human antibody (HAHA) response will bedetected in a transgenic mouse, the method comprising the steps ofattaching an antigenic substance to a fully human or humanized antibodyand administering the combined antigenic substance and antibody to atransgenic mouse that is capable of producing a fully human or humanizedantibody to determine if the combination induces a HAHA response. Insome embodiments, the transgenic mouse comprises human immunoglobulin Vgenes and lacks mouse immunoglobulin V genes. In some embodiments, thetransgenic mouse comprises a set of V genes that essentially consists ofa same set of V genes that a host that is to receive the fully human orhumanized antibody has.

In some aspects, the invention is a transgenic mouse for screening foragents that inhibit the induction of a human anti-human antibody (HAHA)response. The transgenic mouse for screening for agents comprises ahuman gene configured to allow the transgenic mouse to produce a fullyhuman or humanized antibody and a HAHA inducing antibody in thetransgenic mouse. The HAHA inducing antibody is encoded by a gene thatthe transgenic mouse does not possess in its genome. In someembodiments, the transgenic mouse comprises a candidate HAHA inhibitorthat is inside of said transgenic mouse. In some embodiments, the HAHAinducing antibody is encoded by a high-risk gene selected from the groupconsisting of V_(H)3-9 V_(H)3-13, and V_(H)3-64.

In some aspects, the invention is a method for screening for agents thatinhibit the induction of a human anti-human antibody (HAHA) response.The method comprises administering a HAHA inducing antibody to atransgenic mouse where the transgenic mouse comprises a human geneconfigured to allow the transgenic mouse to produce a fully human orhumanized antibody, administering a candidate HAHA inhibitor to thetransgenic mouse, and observing if a resulting HAHA response isinhibited after an amount of time sufficient to allow for a HAHAresponse.

In some aspects, the invention is a method for monitoring a HAHAresponse. The method comprises providing a first transgenic mouse thatcomprises a human gene configured to allow the first transgenic mouse toproduce a fully human or humanized antibody, administering to the firsttransgenic mouse a first foreign antibody under a first condition, anddetermining a presence of a HAHA response in the first transgenic mouse.In some embodiments, the method further comprises providing a secondtransgenic mouse that comprises a human gene configured to allow thesecond transgenic mouse to produce the fully human or humanizedantibody, administering to the second transgenic mouse a second foreignantibody under a second condition, wherein the first condition and thesecond conditions are a same variable but are different from oneanother, determining a presence of a HAHA response in the secondtransgenic mouse, and comparing the presence of the HAHA response in thefirst transgenic mouse to the presence of the HAHA response in thesecond transgenic mouse, thereby determining which condition results ina greater HAHA response. In some embodiments, the first transgenic mouseand the second transgenic mouse are different mice but produce a samefully human or humanized antibody. In some embodiments, the firstforeign antibody and the second foreign antibody have a same amino acidsequence or primary structure. In some embodiments, the first conditionis a first expression system and the second condition is a secondexpression system. In some embodiments, the first condition is a firstformulation and the second condition is a second formulation, whereinthe first formulation is used to produce the first foreign antibody andthe second formulation is used to produce the second foreign antibody.In some embodiments, the first condition is a first degree ofaggregation of the first foreign antibody and the second condition is asecond degree of aggregation of the second foreign antibody. In someembodiments, the first condition is a first amount of the first foreignantibody and the second condition is a second amount of the secondantibody. In some embodiments, the first condition is a first dosingregimen and the second condition is a second dosing regimen. In someembodiments, the first condition is a first route of administration andthe second condition is a second route of administration. In someembodiments, the first and second routes of administration are differentand are selected from the group consisting of: subcutaneously,intravenously, intraperitoneally, intracranially, intradermally,intramuscularly, and orally. In some embodiments, the first condition isa first immune system and the second condition is a second immunesystem, wherein the first and second immune systems have differingamounts of activity. In some embodiments, the first condition is a firstisotype of the first foreign antibody and the second condition is asecond isotype of the second foreign antibody. In some embodiments, thefirst and second foreign antibodies comprise a protein section encodedby a gene that is not expressed in the transgenic animal. In someembodiments, determining a presence of a HAHA response is a qualitativedetermination. In some embodiments, determining a presence of a HAHAresponse is a quantitative determination.

In some aspects, the invention is a method for increasing theprobability that a human anti-human antibody (HAHA) response will bedetected in a transgenic mouse. The method comprises the steps ofattaching an antigenic substance to a fully human or humanized antibody,and administering the combined antigenic substance and antibody to atransgenic mouse that is capable of producing a fully human or humanizedantibody to determine if the combination induces a HAHA response.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a representation of an example of the raw data for anormalized host V gene profile. Host 1 has V genes for genes a-d.

FIG. 1B is a representation of an example of the raw data for anormalized host V gene profile, in a polypeptide or mRNA expressionformat. Host 1 only transcribes or translates genes A, C, and D.

FIG. 1C is a table depicting the frequency of occurrence for variousgenes in the population of hosts 1-5, as a function of the frequency ofthe gene's appearance.

FIG. 1D is a table depicting the frequency of occurrence for variousgenes in the same population of hosts 1-5, as a function of thefrequency of the protein or mRNA appearance.

FIG. 2A is a representation of a series of antibody V genes compared toa profile.

FIG. 2B is a representation of a series of antibody V amino acidsequences compared to a profile.

FIG. 2C is a representation of antibody V region polypeptide structurescompared to a profile.

FIG. 3 depicts a flow chart for a method for determining if a gene is ahigh-risk gene for inducing a HAHA response, and then altering the geneto decrease the risk.

FIG. 4A depicts a flow chart for one method for optimizing an antibody.

FIG. 4B depicts a flow chart for another method for optimizing anantibody.

FIG. 4C depicts a flow chart for another method for optimizing anantibody.

FIG. 5 depicts a flow chart for various methods of selecting andmodifying genes to reduce an antibody's risk of inducing a HAHAresponse. These methods can be used to alter individual antibodies, forexample by removing a V_(H) gene or a V_(L) gene, or to alter thegenomes of HAHA customized XenoMouse® or other transgenic mice, forexample by removing a high-risk gene from the genome. Similarly,antibody display libraries using a fixed number of V region frameworkscan be pre-selected to use only V region frameworks from V genesassessed to have a low probability of eliciting HAHA.

FIG. 6 depicts a representation of a method for determining which andhow a gene can be changed to avoid the loss of functionality but reducethe risk of a HAHA response.

FIG. 7 depicts a representation of a method for determining which andhow an amino acid can be changed to avoid the loss of functionality butreduce the risk of a HAHA response.

FIG. 8 depicts a table with a listing of some of the relevant genes ofthe present embodiments. The table also identifies genes that arerelatively rare, or possible high-risk genes.

FIG. 9 depicts a flow chart of one method by which experimental data canbe used to determine the risk values of genes.

FIG. 10 is a bargraph depicting the presence of V_(H)3-9 in variouscells.

FIG. 11A is a graph displaying the level of immunogenicity induced byantibody A when administered to a XenoMouse® animal via base of tailfollowed by intraperitoneal route in the presence of adjuvant(“BIP/ADJ”).

FIG. 11B is a graph displaying the level of immunogenicity induced byantibody A when administered to a XenoMouse® animal subcutaneously.

FIG. 11C is a graph displaying the level of immunogenicity induced byantibody A when administered to a XenoMouse® animal intravenously.

FIG. 12A is a graph displaying the level of immunogenicity induced byantibody B when administered to a XenoMouse® animal via a BIP/ADJ route.

FIG. 12B is a graph displaying the level of immunogenicity induced byantibody B when administered to a XenoMouse® animal subcutaneously.

FIG. 12C is a graph displaying the level of immunogenicity induced byantibody B when administered to a XenoMouse® animal intravenously.

FIG. 12D is a graph displaying the level of immunogenicity induced by apositive control, KLH via the BIP route (left panel) or subcutaneously(right panel)

FIG. 13A is a graph displaying the level of immunogenicity induced by anantibody (“Ab”) administered subcutaneously.

FIG. 13B is a graph displaying the level of immunogenicity induced by asham-conjugated Ab (“Ab-sham”), administered subcutaneously.

FIG. 13C is a graph displaying the level of immunogenicity induced by anAb conjugated to TCE peptide (“Ab-TCE”), administered subcutaneously.

FIG. 13D is a graph displaying the level of immunogenicity induced byTCE peptide administered subcutaneously.

FIG. 14A is a graph displaying the level of immunogenicity induced by anAb administered intravenously.

FIG. 14B is a graph displaying the level of immunogenicity induced by asham-conjugated Ab (“Ab-sham”), administered intravenously.

FIG. 14C is a graph displaying the level of immunogenicity induced by anAb conjugated to TCE peptide (“Ab-TCE”), administered intravenously.

FIG. 14D is a graph displaying the level of immunogenicity induced byTCE peptide, administered intravenously.

FIG. 15 is a graph displaying the level of immunogenicity induced by theAb and an adjuvant, administered via the BIP route.

FIG. 16A is a depiction of an alignment of various V_(H) genes.

FIG. 16B is a depiction of an alignment of various V_(H) genes.

FIG. 17A through FIG. 171 are lists of amino acid sequences of variousV_(H) genes.

FIG. 18A through FIG. 18P are lists of nucleic acid sequences of variousV_(H) genes.

FIG. 19A through FIG. 19G are lists of amino acid sequences of variousV_(kappa) genes.

FIG. 20A through FIG. 20L are lists of nucleic acid sequences of variousV_(kappa) genes.

FIG. 21A through FIG. 21F are lists of amino acid sequences of variousV_(lambda) genes.

FIG. 22A through FIG. 22J are lists of nucleic acid sequences of variousV_(lambda) genes.

FIG. 23A and FIG. 23B are lists of additional sequences used herein.

DETAILED DESCRIPTION OF THE EMBODIMENTS

It has been discovered that antibody genes that are under-represented inthe general population or in a host can contribute to the immunogenicityof a therapeutic antibody encoded by such a gene (e.g., a HAHAresponse). Identification and characterization of these genes will aidin the assessment of therapeutic candidate monoclonal antibodies and canbe incorporated as a risk factor at the time of preclinical development.This information can be used, not only to estimate the risk that a HAHAresponse will occur, but in the creation of antibodies as well. Thus,antibodies with a reduced risk of inducing a human anti-human antibody(HAHA) response can readily be created and identified.

In general, there are two levels at which the risk that an antibody willinduce a HAHA response can be measured: in an individual and across apopulation (although, in some embodiments, a population can be apopulation of one).

In one aspect, an antibody with a high-risk of inducing a HAHA responsein a particular patient or host can be identified by comparing thegene(s) that can encode the particular antibody (e.g., an antibody geneset) with the genes in the patient or host (e.g., a host gene set). Ifthe patient or host has the same genes as the genes encoding theantibody and if the genes are expressed in the host, then the antibodycan be a low risk antibody. If the host lacks the genes or the proteinencoded by the antibody genes, then there is a risk of a HAHA responseoccurring in the host with the particular antibody. Thus, it would bepossible to pre-screen patients prior to administering an antibody so asto reduce the chance of a HAHA response that would negate the drug'sefficacy or perhaps cause a serious or life-threatening allergicreaction.

In one aspect, the risk that an antibody will induce a HAHA response inan individual in a population can be determined. At the populationlevel, the frequency of the gene in the population can be used to assessthe risk that an individual in the population will experience a HAHAresponse if given the antibody. Thus, the methods and compositions canbe used, on various levels, to gauge the risk of a HAHA response.

Some embodiments relate to methods and systems for reducing the riskthat an antibody will induce a human anti-human antibody (HAHA) responsein an individual.

In one aspect, the embodiments include a method for optimizing anantibody so that it has a reduced risk of inducing a HAHA response in apatient or population of patients. In this method, the antibody isoptimized by identifying whether the antibody was encoded by one or more“risk genes” within the immunoglobulin gene family. If so, then the DNAor protein encoded by the risk gene is altered to reduce the likelihoodthat the antibody will induce a human anti-human antibody (HAHA)response when administered to a patient.

In one embodiment, the risk gene encodes at least a portion of thevariable domain of the antibody. More preferably, the risk gene encodesan immunoglobulin variable (“V”) region of either the heavy chain or thelight chain of the antibody. The risk gene can encode a heavy V gene(V_(H)) gene. More preferably yet, the risk gene encodes a V_(H)3 gene.In another embodiment, the risk gene is a D or a J gene. In anotherembodiment, the risk gene is a light V gene (V_(L)) or a J gene.Preferably, the risk gene is selected from the following: V_(H)3-9,V_(H)3-13, and V_(H)3-64.

Some embodiments of the invention relate to the discovery thatantibodies encoded from certain V family genes are more likely to inducea HAHA response than antibodies encoded from other V genes. Embodimentsfurther relate to the discovery that it is possible to identify if aprotein section from a V gene is likely to cause a HAHA response. Thiscan be determined by examining how common the V gene is in theindividuals of a population to which an antibody containing a portion ofprotein encoded by the V gene will be administered. V genes, and theproteins encoded by them, that appear throughout a population are notvery likely to cause a HAHA response. On the other hand, V genes andtheir product proteins that are rare in the population have a greaterrisk of causing a HAHA response.

Because each antibody contains portions encoded by particular members ofthe V gene family, it is possible to screen for antibodies that wereencoded by specific V genes. Accordingly, if an antibody is known to beencoded from an immunogenic (high-risk) V gene for a particularpopulation or individual, it can be removed as a potential therapeuticantibody. Similarly, if the antibody is encoded by a V gene that isknown to not be immunogenic (low-risk) for a particular population, itcan be selected as a proper candidate for antibody therapy. This canallow one to assemble large pools of different antibodies, each antibodywith a desired function and each antibody with a low risk of inducing aHAHA response. By being able to select or identify which antibodies havea relatively high-risk of inducing a HAHA response in a population, andeliminating those from the pool of antibodies, one can create and uselarge numbers of diverse antibodies, without as much concern over a HAHAresponse occurring.

In another aspect, the method involves the selection of particularlow-risk genes (genes which appear with a frequency greater than aminimum frequency of occurrence or genes that are present in theindividual). The antibody products from these genes are then assumed tohave a low-risk of inducing a HAHA response in a patient. It should berealized that the selection of low risk genes or elimination of highrisk genes applies not only to embodiments concerning the DNA, but alsoembodiments concerning proteins encoded by the genes. Thus, this aspectalso includes a method for selecting low risk antibodies that areencoded by low risk genes.

In another aspect, the method is directed towards selecting an antibodyfor a particular patient to minimize the risk that the antibody willinduce a HAHA response in that patient. In this aspect, for example, theset of genes encoding the antibody (antibody gene set) to beadministered to the patient is compared to genomic, expressed, orcombination of both, antibody gene information of the patient (host geneset). In this manner, one can select an antibody to administer that isencoded by genes known to be present in the patient's genome orexpressed antibody repertoire. In one embodiment, the patient is firstanalyzed to determine the presence of V genes that are used to encodeantibodies for that patient. If, for example, the V_(H)3-13 gene is usedto encode antibodies in the patient, then an antibody utilizing thatV_(H) gene can be administered, as it does not have a high risk ofcausing a HAHA response in that patient. This can be achieved throughsequence or gene comparisons, or through the use of correlative databetween V gene usage and ethnic background, for example.

In another aspect, the method is directed to determining the likelihoodof a HAHA response occurring in order to allow further customization andanalysis of a patient's condition. Knowing this probability will allowthe patient and the care provider to make a more educated guess aboutthe cost benefit analysis of using the antibody. It will also provideadditional information about future possible problems and allow thepatient to start HAHA preventative treatments before any adverse sideeffects but when such side effects are likely.

In another aspect, a method for creating low risk antibodies throughcustomized systems, such as an animal system or an antibody displaysystem, is provided. For example, a XenoMouse® mouse can be created thathas the same gene profile or gene set as a particular population that isidentified. Similarly, antibody display libraries using a fixed numberof V region frameworks can be pre-selected to use only V regionframeworks from V genes assessed to have a low probability of elicitingHAHA. Thus, any antibodies generated from this customized animal ordisplay library will be from those genes that are low risk genes forthat population, thereby creating antibodies with desiredcharacteristics that are encoded by genes that are not high-risk genes.

In another aspect, the XenoMouse® mouse, or customized version thereof,can be used to determine if an antibody will induce a HAHA response. Asthe XenoMouse® mouse can have the same gene set as a potential host (orhost population), by administering the candidate antibody to theXenoMouse® mouse and observing if there is a HAHA response, one candetermine if such a response will occur in the host.

In some embodiments, the XenoMouse® mouse has candidate antibodiesadministered to it and the mouse can then be observed to determine ifthe antibody induced a HAHA response. In some embodiments, additionalsubstances (e.g., candidate HAHA inhibitors) are added to the mouseafter, during, or before the administration of the antibody to determineif the additional substance can reduce or prevent the HAHA response.Thus, compositions and methods for identifying inhibitors of a HAHAresponse are also provided.

In some aspects, the XenoMouse® mouse, or similar transgenic organism,is used to determine if variations in the conditions of a treatment orexperiment contribute to the immunogenicity of the antibody that isadministered to the mouse. Variations of the conditions can include bothvariations on the substances used and variations on how the materialsare used. For example, the amount, frequency of administration, type ofconstant domain for the antibody, or method of administration of theantibody can be varied and the resulting change in HAHA response, ifany, observed.

In another aspect, various databases of information that are useful indetermining or selecting various genes for the optimized antibodies arecontemplated. There are several broad types of databases that arecontemplated herein. There are normalized host V gene profiles thatprovide a general concept of the frequency of occurrence of a gene inthe population. In a preferred embodiment, the V genes are heavy chain Vgenes or light chain V genes. Other databases include or are directed toD genes and J genes. In a preferred embodiment, the frequency ofoccurrence is used to predict and assign a risk value to each gene. Thelower the frequency of occurrence, the higher the risk of a HAHAresponse in a population.

The risk can be correlated to a population in various ways. In oneembodiment, the frequency of a gene is correlated to the ethnicbackground of an individual. In another embodiment, the frequency of agene is correlated to the risk that the individual will have a HAHAresponse, based on the past history of the patient. Such a database canbe produced by comparing antibodies with known sets of genes to thefrequency of occurrence that the antibodies induced a HAHA response in apopulation of patients; thus, methods of creating these databases arealso contemplated. Furthermore, additional information may be stored orexamined in these databases, such as correlations by gene clustering,predicted protein sequences, predicted protein structures, necessarygene arrangement for binding, and predicted effects of altering thegenes. These databases can be useful for determining both high frequency(low risk of inducing a HAHA response) genes and low frequency(high-risk of inducing a HAHA response) genes.

There are also compositions of various optimized antibodies that exhibita reduced likelihood of inducing a human anti-human antibody response.These compositions comprise both amino acid and nucleic acidgene-optimized antibodies that can be substantially pure. Antibodiesproduced, optimized, or selected by the methods described herein arealso contemplated. Gene-optimized antibodies containing only common orlow-risk genes are also contemplated, as are gene-optimized antibodies,which have rare genes in the selection of V genes, wherein the raregenes are not functionally expressed as protein. Variants of thesegene-optimized antibodies are also contemplated.

In one embodiment, a “gene-optimized” or “low-risk” antibody is createdfrom an antibody gene set or gene pool by selecting a gene that iscommon in a host gene profile or a normalized host gene profile. In apreferred embodiment, the gene is a V gene and the host gene profile isa host V gene profile. In one embodiment, the gene-optimized antibody iscreated from the XenoMouse® mouse or customized version thereof.

In one aspect, any of the methods or compositions disclosed herein arecontemplated for use not only for reducing the risk of a HAHA response,but also for reducing the risk of immunogenicity in general. In a morepreferred embodiment, the methods and compositions disclosed herein arecontemplated for reducing the risk of immunogenicity for humanizedantibodies and chimeric and nonhuman antibodies.

In another aspect, the methods and compositions that are to be used inlowering the risk of a HAHA response can also be used to increase therisk of a HAHA response.

In another embodiment, D genes and J genes of the heavy chain and J andV genes of the light chain, as well as combinations of the genes can beanalyzed and used as the V_(H) genes can be used in the presentdisclosure.

I. DEFINITIONS

A HAHA response is an immunogenic response of a human host, orequivalent thereof, to a human part of an antibody. The antibody can bea fully human antibody from any source, for example, an antibody createdby a human or a XenoMouse® mouse. The antibody can also be one that ishumanized, as long as some part of the antibody is human. In someembodiments, the equivalent of a human host is a XenoMouse® mouse. Thus,a XenoMouse® mouse can have a HAHA response to, for example,administered human antibodies, humanized antibodies, antibodies thatcontain human protein sequence or are encoded by human nucleic acidsequence, or XenoMouse® mouse antibodies.

While the HAHA response is most likely induced by the proteins encodedby the risk genes; much of the analysis and data gathering may involvecomparisons at the genetic level. Because of this, and for ease ofdisclosure, the terms “V gene,” “high-risk gene,” and “low-risk gene”can be used to describe not only the genetic material, but also theprotein encoded by the genetic material. Thus, for the purposes of thisspecification, an antibody protein can “have or comprise a high-riskgene.” This is not meant to suggest that an antibody protein is attachedto a piece of DNA. Rather, it refers to the fact that the antibodycontains protein sections that can be encoded by those genes. Since onefeature of many of the present embodiments is the correlation betweengenes (or proteins encoded by the genes) and HAHA risk, it is convenientto discuss these structures generically in terms of units of genes,regardless of whether the actual gene is described or the protein isbeing discussed. Thus, an “antibody that has a V_(H)3-9 gene” actuallymeans that there is a section of protein in the antibody that is encodedby a V_(H)3-9 gene. However, this use of the term “gene” only applieswhen it is describing a protein of some sort (e.g., an antibody). Thus,a mouse with a V_(H)3-9 gene or a vector with a V_(H)3-9 gene, unlessotherwise specified, is referring to actual DNA material. Additionally,in some embodiments, the term “risk” can be replaced by the term“probability;” thus, for example, the probability that a HAHA responsewill occur can be determined or genes with a high probability ofinducing a HAHA response identified or manipulated. As will beappreciated by one of skill in the art, the terms can, in somesituations, be used interchangeably.

“Host V gene profile” refers to the set of V genes of a potential hostor patient. The profile has information concerning the types of genes inthe host and may have information concerning the frequency of thosegenes occurring, in protein form or mRNA, in the host. A profile canexist for individuals or for populations. An example of such a profile,the raw data that makes it up, and alternative variations of profiles,is shown in FIG. 1A-D. By comparing a gene in an antibody to the genesin the host V gene profile, one is able to determine if the potentialgene is common in the host, and thus, if the gene is likely to induce aHAHA response. Similarly, if one compares the frequency of usage of thegene in the profile with a gene of an antibody, one is also able todetermine if a HAHA response has a greater probability of occurring.“Host V gene set” can be used interchangeably with the “host V geneprofile” for individuals and simply refers to the set of relevant genesin the host.

“Normalized host V gene profile” refers to a host V gene profile thathas been normalized by some standard. A normalized host V gene profilemay be normalized in many different ways. For example, at a simplelevel, it is normalized across several people to show the frequency thata gene occurs in any given population. A “family” normalized host V geneprofile would involve using the host V gene profiles of the geneticallyrelated members of a family in order to determine which genes occur andwith what frequency for those family members. An ethnic normalized hostV gene profile would compare various members of particular ethnic groupsin order to determine what the average frequency of occurrence forparticular genes are in that particular ethnic group. A “human”normalized host V gene profile would weigh the relative frequencies ofoccurrence without any other defining feature, apart from the fact thatthe host must be human. A normalized host V gene profile allows one todetermine which genes are risk genes and which genes occur with at leasta common frequency of occurrence. As a normalized host V gene profilewill contain genes from different people, the results are tabulated as afrequency of occurrence of each gene in the entire population. Otherprofiles may exist for non-human beings as well. For instance, anormalized host V gene profile could be made for cats, mice, pigs, dogs,horses, etc. Host V gene profiles can be made across species as well.These profiles can be used for any organism that contains a V gene or Vgene functionally equivalent system. The larger the normalized host Vgene profile, the less customized each gene-optimized antibody will be,and thus the more likely the gene-optimized antibody will induce a HAHAresponse. Gene-optimized antibodies selected from these largernormalized profiles can be used to produce “universal gene-optimizedantibodies.” These antibodies can have a reduced risk of inducing a HAHAresponse in the host animals, but can still be used on many differentorganisms or patients with different host V gene profiles. Theseuniversally optimized V gene antibodies genes can be useful when it isnot convenient to determine the V gene profile of the patient to betreated.

In a population, a “risk gene” is a gene, which because of its lowfrequency of occurrence in the normalized host V gene profile, presentsa noticeable risk that its presence in an antibody will induce a HAHAresponse in the patient. In one embodiment, a risk gene is one thatoccurs in a gene profile less than 1% of the time. In anotherembodiment, a gene is considered a risk gene if it occurs with afrequency of about less than 100% in the normalized host V gene profile,for example: 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,15%, 30%, 49%, 50%, 70%, or 99%. This is also referred to as a “highrisk gene.” In one embodiment, a risk gene is any gene that occurs witha frequency of occurrence that is less than a predetermined frequency ofoccurrence. In an individual, a “high risk gene” is a gene that theindividual lacks or does not express protein for. As appreciated by oneof skill in the art, the high-risk gene itself may not be responsiblefor inducing the HAHA response. As used herein, the term “high-riskgene” refers to the fact that a protein encoded by the gene has a highrisk of inducing a HAHA response. However, because of the varioustechniques in which this information is obtained and can be applied, itis often easier and more accurate to simply refer to these genes ashigh-risk genes, rather than the proteins encoded by high-risk genes.

“Low-risk gene” is a gene that does not present a noticeable risk ofinducing a HAHA response. Alternatively, the low-risk gene may actuallyreduce the risk of inducing a HAHA response. In a population, thesegenes can be identified by their common frequency of occurrence in thehost V gene profile. A gene is a low-risk gene if it occurs with afrequency of at least about 1-100%, for example, 1%, 5%, 10%, 15%,20-29%, 30-39%, 40-49%, 50-59%, 60-69%, 70-79%, 80-89%, 90-94%, 95%,99%, and 100%. A low-risk gene can also be called a “common gene.” Inone embodiment, a low-risk gene is a gene that occurs with a greaterfrequency, in a population, than a minimum or predetermined frequency ofoccurrence. In an individual, a low risk gene is a gene that isexpressed by the individual; and thus, there is a low risk of a HAHAresponse. As appreciated by one of skill in the art, the low-risk geneitself may not be responsible for not inducing the HAHA response. Asused herein, the term “low-risk gene” refers to the fact that a proteinencoded by the gene has a low risk of inducing a HAHA response. However,because of the various techniques in which this information is obtainedand can be applied, it is often easier to refer to these as low-riskgenes, rather than the proteins encoded by low risk genes. As will beappreciated by one of skill in the art, a minimum frequency ofoccurrence can vary depending upon the particular situation and desiredtreatment for a patient. One of skill in the art, in light of thepresent disclosure, will be able to readily determine or set the desiredminimum frequency of occurrence (or predetermined frequency ofoccurrence). As above, a “low-risk” or similar term can be replaced withthe term, “low probability”, in some embodiments.

“Antibody gene set” is the set of genes that encode a particularantibody or antibodies. The entire gene set can be “optimized,” whichmeans that the composition of genes in the gene set share a degree ofsimilarity with a possible host gene set (e.g., host V gene profile foran individual or a population). The gene set consists of V, D, J genesfor the heavy chain and V and J genes for the light chain. A gene setcan include all of the above genes as well.

“V gene” is an immunoglobulin variable gene.

The human immunoglobulin V_(H) (variable, heavy chain) germlinerepertoire comprises at least 123 elements (Matsuda et al., J Exp Med.,188:2151-2162 (1998)), with 41 of these representing functional genes.Several studies have addressed polymorphisms and repertoire expression,sometimes in relation to ethnicity, age or gender. These reports reliedon a single or very few donors (Hufnagle et al., Ann N Y Acad Sci.;764:293-295 (1995); Demaison et al., Immunogenetics., 42:342-352 (1995);Wang et al., Clin Immunol., 93:132-142 (1999); Rao et al., Exp ClinImmunogenet., 13:131-138 (1996); Brezinschek et al., J Immunol.,155:190-202 (1995); and Rassenti et al., Ann N Y Acad Sci., 764:463-473(1995)), analyzed leukemia or autoimmune patients (Dijk-Hard et al., JAutoimmun. 12:57-63 (1999); Logtenberg et al., Int Immunol., 1:362-366(1989); Dijk-Hard et al., Immunology, 107:136-144 (2002); Li et al.,Blood, 103:4602-4609 (2004); Messmer et al., Blood, 103:3490-3495(2004); Johnson et al., J Immunol., 158:235-246 (1997)), focused on alimited number of genes (Pramanik et al., Am J Hum Genet., 71:1342-1352(2002); Rao et al., Exp Clin Immunogenet., 13:131-138 (1996); Huang etal., Mol Immunol., 33:553-560 (1996); and Sasso et al., Ann N Y AcadSci., 764:72-73 (1995)), or categorized V_(H) gene use by family(Hufnagle et al., Ann N Y Acad Sci., 764:293-295 (1995); Rassenti etal., Ann N Y Acad Sci., 764:463-473 (1995); Logtenberg et al., IntImmunol., 1:362-366 (1989); Ebeling et al., Int Immunol., 4:313-320(1992); and Feuchtenberger et al., J Immunol Methods, 276:121-127(2003)). In addition to V_(H), various V_(L) (variable, light chain)genes are also contemplated. The V_(L) genes can be encoded by theV_(kappa) or V_(lambda) locus. One of skill in the art, given thepresent disclosure, will be able to apply the current teachings todetermine the various HAHA associated issues regarding the variousV_(lambda) genes. The various sequences of these V genes are included inSEQ ID NOs: 15-266 and a listing of some of the various genesthemselves, for V_(H), V_(lambda), and V_(kappa), are shown in FIG. 8and FIGS. 17A-17I, 18A-18P, 19A-19G, 20A-20L, 21A-21F, and 22A-22J.

“Optimized gene” is a gene that is present in both an antibody (in termsof encoding a part of the antibody), and in the genes of a potentialhost. When the gene is a V gene, it can be optimized if it is in the“normalized host V gene profile,” or if an individual has the V gene aswell. Such genes are generally considered “low-risk” genes, with someexceptions discussed herein. A gene can be optimized if it is identifiedas being a low-risk gene for a host. Alternatively, a gene can beoptimized if, after a comparison of the gene in the antibody and thehost gene profile, the gene is then changed so that the gene moreclosely resembles the genes in the host gene profile. For example, agene of one antibody may not be present in a host gene profile; deletionof this gene will result in it being optimized.

“Gene-optimized antibody” is an antibody that has at least one low-riskgene. A gene-optimized antibody can be made or selected. If a Vgene-optimized antibody is created, then the antibody contains at leastone low-risk gene for a host V gene profile. If a gene-optimizedantibody is selected, the antibody is relatively enriched for low-riskgenes or relatively depleted of high-risk genes. In one embodiment, agene-optimized antibody is an antibody that is selected based onsimilarities defined by the gene set of the antibodies and the genes ofa potential host. In another embodiment, a gene-optimized antibody is anantibody that is engineered or created to contain as few genes that aredifferent from a potential host's V gene profile. In another embodiment,a gene will be optimized if a D gene appears in a host D gene profile.In another embodiment, a gene will be optimized if a J gene appears in ahost J gene profile.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis(2^(nd) Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland, Mass. (1991)), which is incorporated herein by reference.Stereoisomers (e.g., D-amino acids) of the twenty conventional aminoacids, unnatural amino acids such as α-, α-disubstituted amino acids,N-alkyl amino acids, lactic acid, and other unconventional amino acidscan also be suitable components for polypeptides of the presentinvention. Examples of unconventional amino acids include:4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine,1-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine,3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and othersimilar amino acids and imino acids (e.g., 4-hydroxyproline). In thepolypeptide notation used herein, the left-hand direction is the aminoterminal direction and the right-hand direction is the carboxy-terminaldirection, in accordance with standard usage and convention.

Similarly, unless specified otherwise, the left-hand end ofsingle-stranded polynucleotide sequences is the 5′ end; the left-handdirection of double-stranded polynucleotide sequences is referred to asthe 5′ direction. The direction of 5′ to 3′ addition of nascent RNAtranscripts is referred to as the transcription direction; sequenceregions on the DNA strand having the same sequence as the RNA and whichare 5′ to the 5′ end of the RNA transcript are referred to as “upstreamsequences”; sequence regions on the DNA strand having the same sequenceas the RNA and which are 3′ to the 3′ end of the RNA transcript arereferred to as “downstream sequences”.

The term “antibody” or “antibody peptide(s)” refer to an intactantibody, or a binding fragment thereof that competes with the intactantibody for specific binding. Binding fragments are produced byrecombinant DNA techniques, or by enzymatic or chemical cleavage ofintact antibodies. Binding fragments include Fab, Fab′, F(ab′)₂, Fv, andsingle-chain antibodies. Binding fragments also include “single domain”antibodies such as produced from single V_(H) domain display librariesor derived from camelids. An antibody other than a “bispecific” or“bifunctional” antibody is understood to have each of its binding sitesidentical. An antibody substantially inhibits adhesion of a receptor toa counterreceptor when an excess of antibody reduces the quantity ofreceptor bound to counterreceptor by at least about 20%, 40%, 60% or80%, and more, usually greater than about 85% (as measured in an invitro competitive binding assay). Antibodies cannot only block bindingbut can assist in binding and various enzymatic processes. Additionally,antibodies can be made that, alone, can activate receptors as a ligandnormally would.

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of the heavy chaindefines a constant region primarily responsible for effector function.Human light chains are classified as either kappa or lambda lightchains. Heavy chains are classified as either mu, delta, gamma, alpha,or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, andIgE, respectively. Within light and heavy chains, the variable andconstant regions are joined by a “J” region of about 12 or more aminoacids, with the heavy chain also including a “D” region of about 10 moreamino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed.,2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in itsentirety for all purposes). The variable regions of each light/heavychain pair form the antibody-binding site.

Thus, an intact antibody has two to ten binding sites, depending on theantibody's isotype. Except in bifunctional or bispecific antibodies, thebinding sites are identical.

The chains all exhibit the same general structure of relativelyconserved framework regions (FR) joined by three hypervariable regions,also called complementarity determining regions or CDRs. The CDRs fromthe two chains of each pair are aligned by the framework regions,enabling binding to a specific epitope. From N-terminal to C-terminal,both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2,FR3, CDR3 and FR4. The assignment of amino acids to each domain is inaccordance with the definitions of Kabat Sequences of Proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.(1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 196:901-917 (1987);Chothia et al. Nature 342:878-883 (1989).

A bispecific or bifunctional antibody is an artificial hybrid antibodyhaving two different heavy/light chain pairs and two different bindingsites. Bispecific antibodies can be produced by a variety of methodsincluding fusion of hybridomas or linking of Fab fragments. See, e.g.,Songsivilai & Lachmann Clin. Exp. Immunol. 79: 315-321 (1990), Kostelnyet al. J. Immunol. 148:1547-1553 (1992). Production of bispecificantibodies can be a relatively labor intensive process compared withproduction of conventional antibodies and yields and degree of purityare generally lower for bispecific antibodies. Bispecific antibodies donot exist in the form of fragments having a single binding site (e.g.,Fab and Fv).

The heavy chain variable region is comprised of V, D, and J genesegments that are rearranged resulting in the large diversity ofvariable regions. There are multiple genes that may encode each of the Vregions, D regions and J regions in the mammalian genome, any one ofwhich can be combined in a recombination process to encode a matureantibody chain, thus creating more diversity. Similarly, a light chainvariable region is comprised of a combination of a Vκ (or V_(k) orV_(kappa)) or Vλ (or V_(lambda)) gene selected from a large number of Vκand Vλ genes present in the mammalian genome, with any of a number of Jκor Jλ genes, respectively. The heavy chain and light chain variableregions also undergo other alterations, such as the introduction of Nsequences, which further increases the variability and specificity ofthe variable regions. Thus, the V_(H) region is a combination of any oneof several different heavy chain V, D, and J genes, and the V_(L) regionis a combination of any one of several different light chain V and Jgenes which are created and modified by several different mechanisms.

The term “epitope” includes any protein determinant capable of specificbinding to an immunoglobulin or T-cell receptor or otherwise interactingwith a molecule. Epitopic determinants generally consist of chemicallyactive surface groupings of molecules such as amino acids orcarbohydrate or sugar side chains and generally have specificthree-dimensional structural characteristics, as well as specific chargecharacteristics. An epitope may be “linear” or “conformational.” In alinear epitope, all of the points of interaction between the protein andthe interacting molecule (such as an antibody) occur linearly along theprimary amino acid sequence of the protein. In a conformational epitope,the points of interaction occur across amino acid residues on theprotein that are separated from one another. An antibody is said tospecifically bind an antigen when the dissociation constant is ≦1 μM,preferably ≦100 nM and most preferably ≦10 nM. Once a desired epitope onan antigen is determined, it is possible to generate antibodies to thatepitope. Alternatively, during the discovery process, the generation andcharacterization of antibodies may elucidate information about desirableepitopes. From this information, it is then possible to competitivelyscreen antibodies for binding to the same epitope. An approach toachieve this is to conduct cross-competition studies to find antibodiesthat competitively bind with one another; e.g., the antibodies competefor binding to the antigen. A high throughput process for “binning”antibodies based upon their cross-competition is described inInternational Patent Application No. WO 03/48731. As will be appreciatedby one of skill in the art, practically anything to which an antibodycan specifically bind could be an epitope. An epitope can comprise thoseresidues to which the antibody binds. As will be appreciated by one ofskill in the art, the space that is occupied by a residue or side chainthat creates the shape of a molecule helps to determine what an epitopeis. Likewise, any functional groups associated with the epitope, van derWaals interactions, degree of mobility of side chains, etc. can alldetermine what an epitope actually is. Thus, an epitope may also includeenergetic interactions.

The term “paratope” is meant to describe the general structure of abinding region that determines binding to an epitope. This structureinfluences whether or not and in what manner the binding region mightbind to an epitope. Paratope can refer to an antigenic site of anantibody that is responsible for an antibody or fragment thereof, tobind to an antigenic determinant. Paratope also refers to the idiotopeof the antibody, and the complementary determining region (CDR) regionthat binds to the epitope.

The terms “specifically” or “preferentially” binds to, or similar phraseare not meant to denote that the antibody exclusively binds to thatepitope. Rather, what is meant is that the antibody or variant thereof,can bind to that epitope, to a higher degree than the antibody binds toat least one other substance to which the antibody is exposed.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials.

“Mammal” when used herein refers to any animal that is considered amammal. Preferably, the mammal is human.

Digestion of antibodies with the enzyme, papain, results in twoidentical antigen-binding fragments, known also as “Fab” fragments, anda “Fc” fragment, having no antigen-binding activity but having theability to crystallize. Digestion of antibodies with the enzyme, pepsin,results in a F(ab′)₂ fragment in which the two arms of the antibodymolecule remain linked and comprise two-antigen binding sites. A F(ab′)₂fragment has the ability to crosslink antigen.

“Fv” when used herein refers to the minimum fragment of an antibody thatretains both antigen-recognition and antigen-binding sites. Thesefragments can also be considered variants of the antibody.

“Fab” when used herein refers to a fragment of an antibody thatcomprises the constant domain of the light chain and the CH1 domain ofthe heavy chain.

The term “mAb” refers to monoclonal antibody.

“Label” or “labeled” as used herein refers to the addition of adetectable moiety to a polypeptide, for example, a radiolabel,fluorescent label, enzymatic label chemiluminescent labeled or abiotinyl group. Radioisotopes or radionuclides may include ³H, ¹⁴C, ¹⁵N,³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, fluorescent labels may includerhodamine, lanthanide phosphors or FITC and enzymatic labels may includehorseradish peroxidase, β-galactosidase, luciferase, alkalinephosphatase.

The term “pharmaceutical agent or drug” as used herein refers to achemical compound or composition capable of inducing a desiredtherapeutic effect when properly administered to a patient. Otherchemistry terms herein are used according to conventional usage in theart, as exemplified by The McGraw-Hill Dictionary of Chemical Terms(Parker, S., Ed., McGraw-Hill, San Francisco (1985)), (incorporatedherein by reference).

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and preferably asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present. Generally, a substantially purecomposition will comprise more than about 80 percent of allmacromolecular species present in the composition, more preferably morethan about 85%, 90%, 95%, or 99%. Most preferably, the object species ispurified to essential homogeneity (contaminant species cannot bedetected in the composition by conventional detection methods) whereinthe composition consists essentially of a single macromolecular species.

The term “patient” includes human and veterinary subjects.

The term “SLAM® Technology” refers to the “Selected Lymphocyte AntibodyMethod” (Babcook et al., Proc. Natl. Acad. Sci. USA, i93:7843-7848(1996), and Schrader, U.S. Pat. No. 5,627,052, both of which areincorporated by reference in their entirety).

The term “XENOMAX”™ refers to the use of SLAM Technology with XenoMouse®mice (as described below).

Methods of Identifying High Risk Genes

The identification of genes that have a high risk or a low risk ofinducing a HAHA response in a patient, a population, or an individual ina population can be achieved by determining how frequently the geneappears in the patient or population. Regarding populations andindividuals in populations, V_(H) genes that are relatively common in apopulation have a low risk of inducing a HAHA response in a randommember of the population. Genes that are relatively rare in a populationhave a high risk of inducing a HAHA response in a host patient.Similarly, V genes that are present in an individual are low risk genesfor that individual, while V genes that are absent are high risk genesfor the individual. Thus, the determination of whether or not anantibody expressing a particular gene will result in a HAHA response canbe determined with a database containing information on the frequencywith which particular genes are found in a population. In oneembodiment, for a population, the database is a normalized host V geneprofile. By comparing the gene set of an antibody to this normalizedhost V gene profile one is able to predict the presence of high-riskgenes and low-risk genes. The more similar the population is in geneticmakeup to the patient, the better the prediction should be.

As discussed in more detail below, high risk and low risk genes andantibodies encoded thereby can also be determined experimentally, forexample, by administering a candidate antibody to a XenoMouse® mousethat has no more V genes than those of the host or host population. Ifthe XenoMouse® mouse exhibits a HAHA response, then the gene andantibody encoded by the gene will be a high risk gene.

Normalized Host V Gene Profile

A normalized host V gene profile reflects the frequency of a gene in apopulation. There are several alternatives by which this relationshipcan be described. For example, in FIG. 1A, examples of various V genesof five different people are represented. FIG. 1A represents a selectionof genes in a host and is not meant to suggest any relationship inspatial order or other arrangement. From this, or alternatively, insteadof this, one can use protein expression or mRNA for compiling theprofile, as shown in FIG. 1B. In this example, only genes A, C, D, and Eare expressed as mRNA or protein. From the data in FIG. 1A, a frequencyof occurrence can be determined, the results of which are presented inFIG. 1C. Alternatively, one can obtain a frequency of occurrence as afunction of the gene frequency in the patient, in which cases the factthat a gene can appear as zero copies, one copy, or two copies willfurther influence the above analysis.

Additionally, it can be important to consider not only the frequency ofthe genes, but also the frequency of the proteins that are actuallyexpressed. Thus, a “normalized host V protein profile” or “mRNA profile”can be an appropriate means of comparison as well. In a preferredembodiment, it can be a normalized host V mRNA profile that isdetermined. These last two profiles have the added benefit of removingfrom consideration those genes that, while common, simply never producea protein product. This could be important, especially for those genesin a modified antibody that might normally not be expressed, but for themodification of the antibody. Thus, in situations where one is going tomodify an antibody, it can be advantageous to remove any genes that arealso not functionally expressed. The frequency of occurrence for thegenes can be observed in FIG. 1D. Here, as shown in FIG. 1B, only A, C,D, and E are transcribed or translated.

In a preferred embodiment, the mRNA transcribed from each gene isactually the unit that is examined and compared between profiles. Thus,normalized host V mRNA profiles, and uses thereof are contemplatedembodiments. As understood by one of skill in the art, an mRNA profilecan be used anytime one desires a profile that represents if the genewas transcribed, and thus can be more useful than a DNA sequence. Anamino acid sequence will also provide this type of information, and willhave a greater likelihood of having structurally important regionsconserved.

There are at least two levels at which the frequency of the antibodyprotein can be examined. At a normalized level, the frequency of theprotein is that determined by whether or not a protein product for aparticular gene is produced in a given person. At another level, thefrequency of the use of a protein product of a particular gene isexamined within each person. In this situation, if the gene creates aprotein product that is common in the antibodies of that person, then itwill have a very high frequency of occurrence, and thus be a low-riskgene.

The various frequencies generated in FIG. 1 demonstrate that how onecounts the appearance of each gene, as either for an individual or in apopulation, can influence the frequencies generated, and thus the courseto be taken in later steps. Additionally, a comparison of FIG. 1C toFIG. 1D reveals the large possible differences between a nucleic acidbased gene approach and an amino acid (or mRNA) profile approach. Allembodiments directed to normalized host V gene profiles can also becreated as normalized host V protein profiles, and preferably are.

The two levels of analysis can be combined to produce a normalized hostV gene protein profile where both factors are considered in determiningwhat a low or high risk gene is. This combined approach may beespecially useful when there otherwise is a small number of samples inthe profile.

As discussed below, there may also be structural normalized host V geneprofiles (or normalized host V protein structure profiles). Alternativeembodiments will include D and J versions of these profiles.

Comparisons Using Normalized Host V Gene Profiles:

Once one has a host V gene profile, the information can be comparedagainst the gene set of the antibody in question, or future antibody tobe made. While there are many possible ways of comparing sequences, thecomparisons described herein involve comparisons that involve agene-wise comparison for many of the preferred embodiments, althoughhigher levels of comparison are also contemplated for all of theembodiments.

FIG. 2 depicts several different methods of comparing the pieces ofinformation, and how that information can differ depending on how oneanalyzes the information. Starting with FIG. 2A, the A gene is comparedwith the frequency of the genes in the gene profile to reveal that geneA is common in the profile as its frequency of occurrence is 100%. Thiscould also be an mRNA level of analysis.

At a different level of analysis, in FIG. 2B, the protein sequence ofgene A is compared to a protein or mRNA profile that again results in afrequency of occurrence of 100% (as gene A again occurs at a frequencyof 100% in the profile).

Finally, at another level of analysis, in FIG. 2C, the structure of theprotein encoded by gene A is compared to a structure profile to againreveal that the protein structure is very common as its frequency ofoccurrence is 100%.

The next gene, gene B, is also compared using the three differentmethods. At the first level of comparison, in FIG. 2A, gene B would beinterpreted as a low risk gene as it occurs with a high frequency ofoccurrence, 100%, as can be seen in the profile. At an mRNA/amino acidsequence level of analysis, as shown in FIG. 2B, the B gene proteinproduct is not present in the population. Thus, the B gene can beclassified as a high-risk gene. Finally, at the protein structure level,in FIG. 2C, the B gene displays a structure, if it had been created,that is not present in the structure profile; therefore, gene B would beconsidered a high-risk gene. Thus, this can be considered a high-riskgene.

The next gene, gene C, is also compared using the different methods. InFIG. 2A, gene C could be interpreted as a possible high-risk gene as itsfrequency of occurrence is only 10% in the gene profile. In FIG. 2B,gene C could once again be interpreted as a possible high-risk gene asits frequency of occurrence is still only 10% in the mRNA/proteinprofile. However, in FIG. 2C, the structure of the protein product ofgene C is a very common structure in the structure profile (e.g., 100%);thus, gene C is a low risk gene.

All of the profiles and comparisons described above can also be doneusing mRNA.

Not all of the comparisons need to be gene-wise comparisons. Forinstance, in situations in which one knows a particular gene that is arisk gene, one can further refine the usefulness of the antibodies byattempting a nucleic acid wise optimization within that particular gene.Thus, if one were interested in optimizing an antibody so that theV_(H)3 genes presented less of a risk of inducing a HAHA response, onecould start off optimization by trying to alter the sequence of theparticular V_(H)3 gene to resemble another gene. Thus, normalized host Vsequence profiles are contemplated where sequences are to be comparedrather than entire genes or protein segments.

It could be that following a gene wise comparison and optimization, anadditional level of optimization is desired, at which point theparticular sequences of the genes can be compared in order to furtherreduce aspects of the antibodies that may induce a HAHA response. It isimportant to understand that the sequences are still compared withineach of the genes of the gene set, e.g., V genes, V_(H) genes, V_(H)3genes, or the V_(H)3-9 gene. The risk that is associated with the gene'sfrequency of occurrence can be correlated to the particular structureencoded by the gene.

A gene, for use as described herein, can mean the coding and noncodingaspects of the piece of DNA. Alternatively, rather than looking at theentire gene, only the coding sections can be compared to the potentialhost's V gene background.

Additionally, structural motifs or the entire structure of the antibodycan be compared at the protein level. As many of these gene sections arevery short, the amino acid sequences can be modeled in silico easily andaccurately.

A structure can be generated through homology modeling, aided with acommercial package, such as Insight II modeling package from Accelrys(San Diego, Calif.). Briefly, one can use the sequence of the antibodyto be examined to search against a database of proteins of knownstructures, such as the Protein Data Bank. After one identifieshomologous proteins with known structures, these homologous proteins areused as modeling templates. Each of the possible templates can bealigned, thus producing structure based sequence alignments among thetemplates. The sequence of the antibody with the unknown structure canthen be aligned with these templates to generate a molecular model forthe antibody with the unknown structure. As will be appreciated by oneof skill in the art, there are many alternative methods for generatingsuch structures in silico, any of which can be used. For instance, aprocess similar to the one described in Hardman et al., issued U.S. Pat.No. 5,958,708 (incorporated by reference in its entirety) employingQUANTA (Polygen Corp., Waltham, Mass.) and CHARM (Brooks, B. R.,Bruccoleri, R. E., Olafson, B. D., States, D. J., Swaminathan, S. andKarplus, M., 1983, J. Comp. Chem., 4:187) can be used.

Alternatively, traditional structural examination approaches can beused, such as NMR or x-ray crystallography. These approaches can examinethe structure of a paratope alone, or while it is bound to an epitope.Alternatively, these approaches may be used to look at the individualprotein segments that are each encoded by particular V genes.

These structural motifs are again divided or defined by the actualgenes, even though, as they are incorporated into an antibody, theywould be connected to other amino acids. While the comparisons used tomatch an Ab protein section and a protein section in a profile areprotein structures, the important element of the protein sectionexamined is the gene and the risk associated with that gene, asdetermined by its relative frequency of occurrence. The comparison canbe performed on many different levels. For example, at a relativelysimple level, the primary factor in the comparison can be theside-chains of each amino acid in the chain. Thus, the space-fillingmodel of the side chains of a protein encoded by a V gene can becompared to a similar space-filling model of the protein encoded by theV gene types of the patient. A more complicated comparison would involvethe use of electrostatic interactions of each amino acid in the proteinencoded by the gene, for example. Another method of comparison involvesthe technique taught by U.S. Pub. No. 20040005630 (Published Jan. 8,2004, to Studnicka). This describes a process whereby individual aminoacid positions are compared and particular solvent-exposed side chainsare altered. While the current embodiments are still directed toantibody optimization through gene manipulation, rather than a residueby residue approach, this process can still be useful in altering theresidues in the high-risk gene, once the high-risk gene has beenidentified by the methods described herein. The level of modeling forthe comparison can be increased as much as desired. For example, theproteins encoded by the genes could be modeled as an entire threedimensional protein structure of the antibody, again with the genesbeing used to identify the sections to be compared between the possibleantibody and the gene make up or background of the patient. Thestructural interactions between each of the protein segments encoded bythe V gene segments and other protein in the antibody can also be usedto compare and contrast potential antibodies, or genes of the potentialantibodies, to the set of genes of the patient. By using thiscomparison, a database of protein structures is used to determine therisk that a particular gene structure will induce a HAHA response.

Alternatively, if one wishes to emphasize the similarity of the genesthemselves, then the comparison can be performed on the nucleic acidlevel.

In one embodiment, clusters of particular genes within a gene set areimportant in determining which genes are high-risk genes and which genesare low-risk genes for a particular antibody in a particular patient. Insuch an embodiment, a particular V gene may be a low-risk gene when thegene is compared to a simple normalized host V gene profile; however,when the particular V gene is paired with another gene (e.g. a D or Jgene, or a pairing of a particular V_(H) gene with a particular V_(L)gene), which may also, independently, be a low-risk gene, thecombination results in a cluster of genes which is a high risk cluster.It is the particular combination of two or more common genes that is anuncommon combination, and thus has a high risk, combination. Thus, anantibody that contains such a cluster could have a substantiallikelihood of inducing a HAHA response in the patient, even though asimple, non-cluster, analysis would suggest that all of the genes werelow-risk genes. Whether or not a gene cluster is a high-risk or low-riskcluster is determined and defined in the same way as these terms areused for high-risk and low-risk genes. A cluster can consist of two ormore genes that constitute an antibody.

Another general issue to consider in comparing the frequency ofoccurrence of one gene compared to a gene in the profile is whether ornot to consider the exact sequence of one gene as the only way that thegene can be described or if there is more than one sequence for a gene,that is, are there variants for the genes. There are times where it maybe advantageous to include variants, as they allow a compression ofdata. However, in some instances, especially where, even though verysimilar in terms of sequence, there is a difference in terms offrequency of occurrence between the genes, it would then be detrimentalto use the concept of variants in the analysis.

The term “variant” as used herein, is a polypeptide, polynucleotide, ormolecule that differs from the recited polypeptide or polynucleotide,but only such that the activity of the protein is not detrimentallyaltered unless specified. There can be variants of antibodies, both atthe protein level and at the nucleic acid level. In a preferredembodiment, the ability of a protein variant to function is notdetrimentally altered. In another embodiment, the protein variant canfunction with 10-500% or more of the ability of the wild type mAb. Forexample, the protein variant can function with 10%, 50%, 110%, 500%, orgreater than 500% of the ability of the wild type mAb. In oneembodiment, the range of functional abilities between 10-500% isincluded. In one embodiment, binding ability is the functionality and itcan be reflected in many ways, including, but not limited to the k_(a),k_(d), or K_(D) of the variant to an epitope. In one embodiment,variants of V genes possess the same probability of risk for inducing aHAHA response. Such variants are called “risk constant variants.” In analternative embodiment, variants of V genes possess differentprobabilities for inducing a HAHA response. Such variants may be groupedin “function constant variants,” or more specifically called “riskvariable variants.”

Variant antibodies can differ from the wild-type sequence bysubstitution, deletion, or addition of five amino acids or fewer. Suchvariants can generally be identified by modifying one of the disclosedpolypeptide sequences, and evaluating the binding properties of themodified polypeptide using, for example, the representative proceduresdescribed herein. Polypeptide variants preferably exhibit at least about70%, at least about 90% at least about 95%, or 99% identity to theidentified polypeptides. Preferably, the variant differs only inconservative substitutions and/or modifications. Variant proteinsinclude those that are structurally similar and those that arefunctionally equivalent to other antibody protein structures. In anotherembodiment, the antibody protein is a variant if it is functionallyequivalent to another antibody protein, so long as the paratope of thevariant is similar to the paratopes of the other antibody variant.

In one embodiment, the antibody is a variant if the nucleic acidsequence can selectively hybridize to the wild-type sequence understringent conditions. Suitable moderately stringent conditions includeprewashing in a solution of 5×SSC; 0.5% SDS, 1.0 mM EDTA (pH 8:0);hybridizing at 50° C.-65° C., 5×SSC, overnight or, in the event ofcross-species homology, at 45° C. with 0.5×SSC; followed by washingtwice at 65° C. for 20 minutes with each of 2×, 0.5× and 0.2×SSCcontaining 0.1% SDS. Such hybridizing DNA sequences are also within thescope of this invention, as are nucleotide sequences that, due to codedegeneracy, encode an antibody polypeptide that is encoded by ahybridizing DNA sequence. The term “selectively hybridize” referred toherein means to detectably and specifically bind. Polynucleotides,oligonucleotides, and fragments thereof selectively hybridize to nucleicacid strands under hybridization and wash conditions that minimizeappreciable amounts of detectable binding to nonspecific nucleic acids.High stringency conditions can be used to achieve selectivehybridization conditions as known in the art and discussed herein.Generally, the nucleic acid sequence homology between thepolynucleotides, oligonucleotides, and fragments of the invention and anucleic acid sequence of interest will be at least 80%, and moretypically at least 85-89%, 90-94%, 95-98%, 99%, and 100%. Two amino acidsequences are homologous if there is a partial or complete identitybetween their sequences. For example, 85% homology means that 85% of theamino acids are identical when the two sequences are aligned for maximummatching. Gaps (in either of the two sequences being matched) areallowed in maximizing matching; gap lengths of 5 or less are preferredwith 2 or less being more preferred. Alternatively and preferably, twoprotein sequences (or polypeptide sequences derived from them of atleast 30 amino acids in length) are homologous, as this term is usedherein, if they have an alignment score of at more than 5 (in standarddeviation units) using the program ALIGN with the mutation data matrixand a gap penalty of 6 or greater. See Dayhoff, M. O., in Atlas ofProtein Sequence and Structure, pp. 101-110 (Volume 5, NationalBiomedical Research Foundation (1972)) and Supplement 2 to this volume,pp. 1-10. The two sequences or parts thereof are more preferablyhomologous if their amino acids are greater than or equal to 50%identical when optimally aligned using the ALIGN program. The term“corresponds to” is used herein to mean that a polynucleotide sequenceis homologous (i.e., is identical, not strictly evolutionarily related)to all or a portion of a reference polynucleotide sequence, or that apolypeptide sequence is identical to a reference polypeptide sequence.In contradistinction, the term “complementary to” is used herein to meanthat the complementary sequence is homologous to all or a portion of areference polynucleotide sequence. For illustration, the nucleotidesequence “TATAC” corresponds to a reference sequence “TATAC” and iscomplementary to a reference sequence “GTATA”.

One of skill in the art will realize that, because of the high degree ofsimilarity between many of the V genes, that certain genes may becategorized as variants of the same V gene when they are in factdifferent genes. For the purposes of the present methods andcompositions, even if a V gene is considered a variant of another Vgene, the two genes will not, unless specifically denoted (“variantgene” or “variant gene set”), be considered to be the same gene with anadditive degree of risk.

Variant genes, variant gene sets, and variant host V gene profiles arecontemplated in the present embodiments as a higher level of possibleanalysis. The variant genes, variant gene sets, and variant host V geneprofiles can be used for any of the disclosed embodiments. Oneparticular advantage of such a higher level of analysis is thatcombining the particular genes in this manner allows one to add a littlevariability into each level of analysis. This allows the genes selectedfor to be more acceptable to a larger group of people. In other words,by breaking down the pure gene level of analysis and using a variantgene level of analysis, it is more likely that the genes selected willbe low-risk genes for a larger group of people.

In one embodiment, these closely related variant genes could beconsidered to be the same. In such an embodiment, if one were developinga V gene profile, such variant genes can be considered as a single typeof V gene. In such a situation, this could result in fewer individualgenes being categorized in each profile. However, those genes that werepresent would generally have a higher apparent risk of inducing a HAHAresponse. To the extent that the risk associated with each of the genesrises the same amount, this is not truly indicative of core proteinstructures that are responsible for inducing a HAHA response. However,to the extent that only certain combinations of variant genes have ahigher risk associated with them when the variants are combined acrossgenes, then this is indicative of core structures that probably arerelated to inducing a HAHA response.

In light of this, while the use of variants in defining gene sets maynot always be useful for developing profiles, the variants can be usefulin determining or estimating relevant sections of genes to be altered inorder to alter the risk that a particular gene will induce a HAHAresponse. As an example, one has three different genes of interest, A at10% frequency, B at 100% frequency, and C at 1% frequency in theparticular gene profile, and one wants to optimize gene A. If genes Band C are structural variants of gene A, and genes B and C have adifferent level of risk associated with them than the risk associatedwith gene A, then one can compare the three structures and modify gene Aso that its protein structure will be similar to B, and different to C(see e.g., the discussion below regarding FIG. 6 and FIG. 7). Ideally,the differences between C and B should not alter the functionality ofthe antibody, as all three proteins have the same function, and shouldresult in an antibody with a reduced risk of inducing a HAHA response.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotide or amino acid sequences: “referencesequence”, “comparison window”, “sequence identity”, “percentage ofsequence identity”, and “substantial identity”. A “reference sequence”is a defined sequence used as a basis for a sequence comparison; areference sequence may be a subset of a larger sequence, for example, asa segment of a full-length cDNA or gene sequence given in a sequencelisting or may comprise a complete cDNA or gene sequence. Generally, areference sequence is at least 18 nucleotides or 6 amino acids inlength, frequently at least 24 nucleotides or 8 amino acids in length,and often at least 48 nucleotides or 16 amino acids in length. Since twopolynucleotides or amino acid sequences may each (1) comprise a sequence(i.e., a portion of the complete polynucleotide or amino acid sequence)that is similar between the two molecules, and (2) may further comprisea sequence that is divergent between the two polynucleotides or aminoacid sequences, sequence comparisons between two (or more) molecules aretypically performed by comparing sequences of the two molecules over a“comparison window” to identify and compare local regions of sequencesimilarity. A “comparison window”, as used herein, refers to aconceptual segment of at least 18 contiguous nucleotide positions or 6amino acids wherein a polynucleotide sequence or amino acid sequence maybe compared to a reference sequence of at least 18 contiguousnucleotides or 6 amino acid sequences and wherein the portion of thepolynucleotide sequence in the comparison window can comprise additions,deletions, substitutions, and the like (e.g., gaps) of 20 percent orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by the local homology algorithm of Smith and Waterman Adv.Appl. Math. 2:482 (1981), by the homology alignment algorithm ofNeedleman and Wunsch J. Mol. Biol. 48:443 (1970), by the search forsimilarity method of Pearson and Lipman Proc. Natl. Acad. Sci. (U.S.A.)85:2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage Release 7.0, (Genetics Computer Group, 575 Science Dr., Madison,Wis.), Geneworks, or MacVector software packages), or by inspection, andthe best alignment (i.e., resulting in the highest percentage ofhomology over the comparison window) generated by the various methods isselected.

The term “sequence identity” means that two polynucleotide or amino acidsequences are identical (i.e., on a nucleotide-by-nucleotide orresidue-by-residue basis) over the comparison window. The term“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U, or I) or residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the comparison window (i.e., the windowsize), and multiplying the result by 100 to yield the percentage ofsequence identity. The terms “substantial identity” as used hereindenotes a characteristic of a polynucleotide or amino acid sequence,wherein the polynucleotide or amino acid comprises a sequence that hasat least 85 percent sequence identity, preferably at least 90 to 95percent sequence identity, more usually at least 99 percent sequenceidentity as compared to a reference sequence over a comparison window ofat least 18 nucleotide (6 amino acid) positions, frequently over awindow of at least 24-48 nucleotide (8-16 amino acid) positions, whereinthe percentage of sequence identity is calculated by comparing thereference sequence to the sequence which can include deletions oradditions which total 20 percent or less of the reference sequence overthe comparison window. The reference sequence can be a subset of alarger sequence. Amino acids or nucleic acids with substantial identityto the wild-type protein or nucleic acid are examples of variants of thewild-type protein or nucleic acid.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, preferably at least 90 percent sequence identity,more preferably at least 95 percent sequence identity, and mostpreferably at least 99 percent sequence identity. Preferably, residuepositions that are not identical differ by conservative amino acidsubstitutions. Conservative amino acid substitutions refer to theinterchangeability of residues having similar side chains. For example,a group of amino acids having aliphatic side chains is glycine, alanine,valine, leucine, and isoleucine; a group of amino acids havingaliphatic-hydroxyl side chains is serine and threonine; a group of aminoacids having amide-containing side chains is asparagine and glutamine; agroup of amino acids having aromatic side chains is phenylalanine,tyrosine, and tryptophan; a group of amino acids having basic sidechains is lysine, arginine, and histidine; and a group of amino acidshaving sulfur-containing side chains is cysteine and methionine.Preferred conservative amino acids substitution groups arevaline-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamic-aspartic, and asparagine-glutamine.Polypeptides with substantial identity can be variants.

Variant proteins also include proteins with minor variations. Asdiscussed herein, minor variations in the amino acid sequences ofantibodies or immunoglobulin molecules are contemplated as beingencompassed by the present invention, providing that the variations inthe amino acid sequence maintain at least 75%, between 75-99% identity,for example, 80-89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and99% identity. In another embodiment the variants are all encoded by agene located at a particular chromosomal address. In particular,conservative amino acid replacements are contemplated. Conservativereplacements are those that take place within a family of amino acidsthat are related in their side chains. Genetically encoded amino acidsare generally divided into families: (1) acidic=aspartate, glutamate;(2) basic=lysine, arginine, histidine; (3) non-polar=alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and(4) uncharged polar=glycine, asparagine, glutamine, cysteine, serine,threonine, tyrosine. More preferred families are: serine and threonineare aliphatic-hydroxy family; asparagine and glutamine are anamide-containing family; alanine, valine, leucine, and isoleucine is analiphatic family; and phenylalanine, tryptophan, and tyrosine are anaromatic family. For example, it is reasonable to expect that anisolated replacement of a leucine with an isoleucine or valine, anaspartate with a glutamate, a threonine with a serine, or a similarreplacement of an amino acid with a structurally related amino acid willnot have a major effect on the binding or properties of the resultingmolecule, especially if the replacement does not involve an amino acidwithin a framework site. Whether an amino acid change results in afunctional peptide can readily be determined by assaying the specificactivity of the polypeptide derivative. Such assays are described indetail herein. Fragments or analogs of antibodies or immunoglobulinmolecules can be readily prepared by those of ordinary skill in the art.Preferred amino- and carboxy-termini of fragments or analogs occur nearboundaries of functional domains. Structural and functional domains canbe identified by comparison of the nucleotide and/or amino acid sequencedata to public or proprietary sequence databases. Preferably,computerized comparison methods are used to identify sequence motifs orpredicted protein conformation domains that occur in other proteins ofknown structure and/or function. Methods to identify protein sequencesthat fold into a known three-dimensional structure are known. Bowie etal. Science 253:164 (1991). Thus, the foregoing examples demonstratethat those of skill in the art can recognize sequence motifs andstructural conformations that may be used to define structural andfunctional domains in accordance with the antibodies described herein.

Preferred amino acid substitutions are those which: (1) reduce the riskof the induction of a HAHA response to the protein (2) increase thestructural similarity of a peptide with a risk of inducing a HAHAresponse to a peptide with a lower risk of inducing a HAHA response (3)reduce susceptibility to proteolysis, (4) reduce susceptibility tooxidation, (5) alter binding affinity for forming protein complexes, (6)alter binding affinities, and (7) confer or modify other physicochemicalor functional properties of such analogs. Analogs or variants caninclude various muteins of a sequence other than the naturally occurringpeptide sequence. For example, single or multiple amino acidsubstitutions (preferably conservative amino acid substitutions) may bemade in the naturally occurring sequence (preferably in the portion ofthe polypeptide outside the domain(s) forming intermolecular contacts. Aconservative amino acid substitution should not substantially change thestructural characteristics of the parent sequence (e.g., a replacementamino acid should not tend to break a helix that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles (Creighton, Ed., W. H. Freeman andCompany, New York (1984)); Introduction to Protein Structure (C. Brandenand J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); andThornton et al. Nature 354:105 (1991), which are each incorporatedherein by reference.

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino-terminal and/or carboxy-terminal deletion, but wherethe remaining amino acid sequence is identical to the correspondingpositions in the naturally-occurring sequence deduced, for example, froma full-length cDNA sequence. Fragments typically are at least 5-70 aminoacids long, and include fragments that are at least 5, 6, 8 or 10 aminoacids long, preferably at least 14 amino acids long, more preferably atleast 20 amino acids long, usually at least 50 amino acids long, andeven more preferably at least 70 amino acids long. The term “analog” asused herein refers to polypeptides which are comprised of a segment ofat least 25 amino acids that has substantial identity to a portion of adeduced amino acid sequence. Analogs typically are at least 20 aminoacids long, preferably at least 50 amino acids long or longer, and canoften be as long as a full-length naturally-occurring polypeptide. Bothfragments and analogs are forms of variants

Peptide analogs are commonly used in the pharmaceutical industry asnon-peptide drugs with properties analogous to those of the templatepeptide. These types of non-peptide compound are termed “peptidemimetics” or “peptidomimetics”. Fauchere, J. Adv. Drug Res. 15:29(1986); Veber and Freidinger TINS p. 392 (1985); and Evans et al. J.Med. Chem. 30:1229 (1987), which are incorporated herein by reference.Such compounds are often developed with the aid of computerizedmolecular modeling. Peptide mimetics that are structurally similar totherapeutically useful peptides can be used to produce an equivalenttherapeutic or prophylactic effect. Generally, peptidomimetics arestructurally similar to a paradigm polypeptide (i.e., a polypeptide thathas a biochemical property or pharmacological activity), such as humanantibody, but have one or more peptide linkages optionally replaced by alinkage selected from the group consisting of: —CH₂NH—, —CH₂S—,—CH₂—CH₂—, —CH═CH—(cis and trans), —COCH₂—, —CH(OH)CH₂—, and —CH₂SO—, bymethods well known in the art. Systematic substitution of one or moreamino acids of a consensus sequence with a D-amino acid of the same type(e.g., D-lysine in place of L-lysine) can be used to generate morestable peptides. In addition, constrained peptides comprising aconsensus sequence or a substantially identical consensus sequencevariation can be generated by methods known in the art (Rizo andGierasch Ann. Rev. Biochem. 61:387 (1992), incorporated herein byreference); for example, by adding internal cysteine residues capable offorming intramolecular disulfide bridges which cyclize the peptide.Peptide mimetics and peptidomimetics are both forms of variants.

As will be appreciated by one of skill in the art, while the abovediscussion focused on comparisons of V genes and with normalized V geneprofiles, these techniques, where appropriate, can also be used tocompare V genes in a candidate antibody to the V genes present in anindividual. Of course, the major difference being that, in this latersituation, risk is defined by the presence or absence of the gene,rather than frequencies in a population.

Methods of Selecting Genes for Creating Gene-Optimized Antibodies.

One embodiment is directed to the method of selecting genes to beexpressed in a gene-optimized antibody. Such a gene-optimized antibodyis a functional antibody that has a reduced likelihood of inducing aHAHA response. How the genes are selected, if they are selected formodification, and what type of modification they receive are possibleissues. These can be addressed by examining the gene set of an antibodyand the frequency of each gene's occurrence in a normalized host V geneprofile or if the gene occurs in an individual's V gene profile. Withthis information, V genes for an antibody (or an antibody itself) can beselected in order to make or obtain a gene-optimized antibody. By“antibody gene set” what is meant is the genetic composition of theantibody. By “host gene set” what is meant is the body of relevantgenes, in this case V genes, which are in the host. In one embodimentthis will include the V gene, the J gene, and the D gene. In anotherembodiment, this includes the V_(H) gene and the V_(L) gene. In anotherembodiment, this includes the V_(H) gene. Of course, the set may alsorefer to the mRNA, protein sequence, or structural versions of each ofthe genes. Additionally, a gene set can refer to the set of genes in thegene profile of the host, a population, or a XenoMouse® mouse. Forexample, these can be denoted as an “antibody V gene set” or a “host Vgene set.” When the host V gene profile is for a single person, it willbe the same as the host V gene set. In other words, while a normalizedprofile can describe the percent of people that have various genes, theV gene set simply denotes which genes they have.

In one embodiment, the selection criterion involves minimizing thenumber of high-risk genes, or variants thereof, and/or a maximizing thenumber of low-risk genes or variants thereof. In another embodiment,genes that occur with a frequency beneath a certain minimum frequency ofoccurrence are removed, as shown in FIG. 3. Alternatively, any high riskgenes or variants that are present can be acceptable, as long as theyare not functional, meaning that they do not produce protein, or theprotein produced is a low-risk variant of the normally high risk geneprotein.

The minimum frequency of occurrence, the frequency of occurrence that isthe boundary between a high-risk and a low-risk gene, can vary dependingupon the effect on the antibody's functionality. In one embodiment, afrequency of occurrence of more than 50% will be considered highfrequency, while a frequency of occurrence of less than 50% will beconsidered low frequency. In another embodiment, a frequency ofoccurrence between 50-100% is considered high frequency (e.g., lowrisk), while a frequency of occurrence of less than 50% will beconsidered low frequency. In another embodiment a frequency ofoccurrence of any of: 0-99, including 0-1, 1, 2, 3, 4-15, 15-30, 30-50,50-70, 70-90, or 90-99 percent can each be considered a low frequency ofoccurrence. In another embodiment, a frequency of occurrence of any of100 or less, including: 100, 100-99, 98, 97, 96-85, 85-50, 50-30, 30-10,or 10-minimal percent could each be considered a high frequency ofoccurrence.

While it may seem counterintuitive that a gene that occurs in 99 percentof the antibodies in a population could be deemed low frequency, theseare relative terms, and the range of these terms varies by thepopulation or subpopulation examined and the intended use. Thus, in avery small population, or in a population that has a very smallselection of V genes, where there is very little variation in the genesused to create the antibodies, it can be that the frequency ofoccurrence is well above 50% for any of the genes. However, this doesnot mean that removal of such a gene would not reduce the likelihoodthat a HAHA response can be induced by an organism creating antibodiesto the antigens.

An additional factor to consider in deciding to alter a particular geneis how rare the gene is. As described in detail below, there are severalalternative ways in which one may characterize a gene as high risk.Additionally, as demonstrated in FIG. 1, the exact frequencies ofoccurrence can vary depending on how the data is analyzed. For example,in an embodiment with a minimum frequency of occurrence, any gene thatis less frequent than the minimum frequency of occurrence will be deemeda high risk gene, such as that shown in FIG. 3. A more in depth analysisis provided by correlating the frequency of the gene to a value thatrepresents the risk associated with the gene. For example, given twopossible high risk genes, if one occurs in a normalized V gene profilewith a frequency of 1% and the other appears with a frequency of 10%,the gene with a frequency of 10% could be ten fold less likely to causea HAHA response in any person in the population. As appreciated by oneof skill in the art, this relationship of risk need not be linear, caninclude certain minimum frequencies of occurrence, and can be determinedin many ways, including experimentally, as described below. Thispercentage-weighted degree of risk allows one to also estimate thedegree of change that may be required in order to avoid the risk geneinducing a HAHA response. For example, a gene that is very rare mayrequire a much larger modification than one that is only slightly rare.While this may not be an absolute indicator of how gene modificationshould occur, when this is combined with sequence comparisons of thehigh-risk gene and low-risk genes, as well as sequence comparisonsbetween genes with similar sequences, but different degrees of risk, oneskilled in the art will be able to determine how large a modificationwould be prudent.

As will be appreciated by one of skill in the art, the relatively simple“high risk” or “low risk” characterization of a gene may not be asadvantageous as more detailed description of the risk associated withthe gene. For example, in some embodiments, the risk associated with thegene can be characterized as a “medium risk.”

Alternatively, the risk can also be described in terms of amount of theV gene in the profile. This can be done across a population, or withinan individual. For example, across a population, the risk can becalculated in various weighted manners, a simple method would be throughpercentages of the people having the gene. For example, a first V genecan be present in 90% of a population, a second V gene present in 99%and a third present in 100%. As such, the first V gene can be describedas having a 10% risk, the second a 1% risk, and the third, no risk.Thus, the risk denotes the chance that a random person from thepopulation will not have the gene and thus treat an antibody expressingthe gene in an adverse manner. Within an individual, the percentage candescribe, the similarity of the protein product of the V gene ofinterest with a protein in the host.

Methods for Creating Gene-Optimized Antibodies.

As will be appreciated by one of skill in the art, there are manyalternatives for creating the antibodies described herein, some ofwhich, without limitation, are described in FIG. 4.

Antibody Modification

The antibodies can be modified as described herein in order to eliminateprotein product of high-risk genes, as in FIG. 4A. For instance, in oneembodiment, a point mutation is used to reduce the risk that the proteinwill induce a HAHA response.

In one embodiment, the high-risk gene is altered so it resembles a lowerrisk gene in certain important aspects, such as protein structure, asdescribed in detail below. This can be advantageous in that relativelyminor changes in the protein structure can easily result in a greatreduction in the risk of a HAHA response being induced. These changes inprotein structure can be achieved and directed by a comparison of thehigh-risk gene and a low-risk gene. One can find the closest low-riskgene that is similar to the high-risk gene and change the high-risk geneso that the protein structure of the high-risk gene resembles theprotein structure of the low-risk gene. Of course, through routineexperimentation and testing, this process can be optimized throughmultiple rounds of comparing sequences of V genes, altering the sequenceof the high-risk gene in the antibody to more closely resemble thelow-risk gene, and then testing the antibody for retention of bindingand activity or its risk of inducing a HAHA response or both.Additionally, multiple gene comparisons of high-risk genes can be usedto determine trends in the high-risk genes; these trends could then beselectively targeted for alteration in order to produce optimizedantibodies.

In another embodiment, the high-risk gene can be replaced by a low-riskgene. The replacement can be similar to the high-risk gene (apart fromthe fact that the new gene should have a lower risk associated with thegene). Alternatively, the replacement gene can be a gene that, on aprotein level, would be structurally similar to the gene it isreplacing. Certain characteristics of the high probability gene thatconfer function such as the CDR3 or somatic mutations can be engineeredonto the framework of the low probability gene in order to retainbinding specificity and functional activity. Alternatively, thereplacement gene can be a gene that possesses an especially good abilityto replace other V genes. Such a gene can be determined through thecourse of the above-described experimental process.

In one embodiment, the gene-optimized antibodies are then tested to seethat they still function as desired.

Antibody Selection

The low risk antibodies can be selected from a group of antibodies basedon the antibodies's lower risk of inducing a HAHA response, as is shownin FIG. 4B. At a simple level, this is done by selecting an antibodythat is minimized for high-risk genes in a normalized host gene profile.Another example by which this can be done, described more fully below,is to determine the ethnic background of a patient and then to select anantibody from a pool of antibodies, wherein the antibody comprises a setof genes that are optimized for the occurrence of genes that are commonin the particular ethnic background of the patient. In a preferredembodiment, the genes examined in the gene set are V genes and theprofile is a normalized host V gene, mRNA, amino acid, or proteinstructure profile.

In one method of selecting an antibody for a patient, the gene sets inthe antibody is compared with the possible genes in the patient. Whenthe genes are similar, this indicates that the type of gene is notlikely to cause a HAHA response in the patient. When this is applied toa population, a weighted degree of risk is associated with each gene;thus, the presence of a very rare gene (e.g. 0.001%) will indicate agreater degree of risk than the presence of only a rare gene (e.g.0.01%). Additionally, any risk associated with particular host V geneprofiles can be considered. For example, if an antibody with aparticular gene set would normally be considered low-risk, based on anormalized host V gene profile, but the gene set has a high risk for oneparticular subset of the population which makes up the normalized host Vgene profile, then whether or not to administer this to an individual inthe population can be questioned. This is especially relevant if theindividual shows any indicators of being part of the high-risk subset ofthe normalized host V gene profile.

One of skill in the art will recognize that this type of comparison isnot a simple base-by-base sequence comparison. Rather, gene sequences,mRNA, protein sequences, or protein structures are compared that aredefined by the boundaries of the gene. Thus, the results from a fullsequence comparison can be different from the results of a gene-by-genecomparison. Because of this, the determination of whether or not theparticular antibody will induce a HAHA response need not be the same inmost situations. This is especially relevant to situations in which onedevelops a profile based on recurring sequences compared to a profilebased on recurring genes. Sequences that are common across all V genes,even those that are high-risk, will show up in a profile as low-riskelements. This could result in sequences that are actually high-riskbeing incorrectly lowered in their risk evaluation if high risk geneshave these common structures. In contrast to this, by correlatingfrequency of occurrence or presence or absence with entire genes, onlythe genes that are high-risk will be classified as high-risk and onlythe genes that are low-risk will be classified as low risk.

While one could simply select genes that are low-risk genes for aparticular patient and then make such an antibody with those genes, theability to create an antibody for each patient may not be desirable orpractical. Additionally, the precise V gene profile of the patient maynot be known, or there may not be enough time to create a novelantibody. Thus, in these situations, selecting an antibody for apatient, based on the patient's assumed V gene profile is a usefulalternative.

In the selection of V gene-optimized antibodies by comparing the genesin a pool of antibodies with an assumed host V gene profile, a largerselection of antibodies comprising various combinations of V genes canbe beneficial in obtaining an antibody with a particular combination ofV genes that is minimized for risk genes. In such a situation, it is notthat having a large number of different V genes is necessarily requiredin obtaining a V gene-optimized antibody, but that having a larger poolof antibodies makes it more likely that one antibody in the pool willcontain few risk genes or many low-risk genes for a given patient.

In one embodiment, the method of selecting an antibody for a patientinvolves a collection of all available antibodies comprising differenttypes of genes in a “pool.” The pool can comprise antibodies withnonhuman V genes, or other nonhuman genes. For example, V genes fromrodents, XenoMouse® mice, or pigs. In one embodiment, these pools may becomputational databases where one can computationally compare thelikelihood of any particular V gene inducing a HAHA response in anyparticular person, group of people, or entire population. The likelihoodis determined as described herein, where commonly occurring V genes havea low likelihood and rare V genes have a high likelihood of inducing aHAHA response. Alternatively the pools could be physically embodied asan actual collection of the various antibodies, or genes thereof. Insome embodiments, these pools may comprise D and J genes, either incombination or as separate pools.

Alternatively, in order to reduce cost and storage space requirements,the pools can consist of “universally gene-optimized antibodies.” Thismeans that the antibodies are selected so that they will reduce the riskof inducing a HAHA response when administered across a large population(e.g., 1000-10,000, 10,000-one million, one million to 100 million, 100million to 6 billion). Thus, while these antibodies may not becompletely optimized to each individual, they will allow a singlegene-optimized antibody (or a single set) to be administered to manydifferent people, with a benefit occurring across the population as awhole.

As will be appreciated by one of skill in the art, it is possible tohave pools that are customized to particular groups of people. This isespecially useful where particular groups of people share common V geneusage. Thus, without knowing the precise host V gene profile, one isable to obtain a better fit of profile to risk, than if one simplyselected possible antibodies based on an entire population host V geneprofile.

In one embodiment, the V gene profile that is created, and thus the poolof genes from which one can create or select an antibody for a patient,is based on ethnic background. For example, there are four differentethnic groups, Northern European, Southern European/African, SouthernAsian, and Northeastern Asia. This particular division is especiallysignificant since data on ethnicity appears to be warranted because ofpreviously published work on the segregation of human IgG1 allotypeswith these four different ethnic origins. (See, for example, Pandey, J.P., Vaccine. 19 (6), 613-617, (2000); Hougs, et al., Tissue Antigens, 61(3), 231-239, (2003); Juul, et al., Tissue Antigens, 49 (6), 595-604,(1997); Atkinson, et al., Immunogenetics, 44 (2), 115-120, (1996)).Similarly, among the two human IgG2 allotypes, one is predominant inhumans with African ethnic background and the other is predominantlyused by the rest of world's population. While allotypes refer to allelicvariations in the sequence of the constant region of antibody chains, itis reasonable that V gene usage also segregates with ethnicity.

The pool of antibodies can comprise any type of antibody. In oneembodiment, the pool comprises human antibodies. In another embodiment,the pool comprises antibodies from any human Ig-transgenic animal. Inanother embodiment, the pool comprises antibodies from a XenoMouse®animal. In another embodiment, the pool of antibodies comprisesgene-optimized antibodies. The gene-optimized antibodies can be any ofthe gene-optimized antibodies disclosed herein. In another embodiment,the pool comprises universally gene-optimized antibodies. In anotherembodiment, the pool comprises a mixture of all of these types ofantibodies.

Additionally, the pools themselves can be optimized according to othercharacteristics of people. For example, there can be a pool that isoptimized for patients with a particular characteristic, such as familymedical history, or for a particular genotype of the patient.

As appreciated by one of skill in the art, the selection process formatching an antibody or gene-optimized antibody to a patient can varyfrom situation to situation. As described above, additional factors,such as the size of the pool and the nature of the pool will come intoplay.

Organisms that Produce Gene-Optimized Antibodies

Transgenic animals can be produced that only or primarily possesslow-risk genes, or at least no genes that are high risk, in order toproduce the optimized protein, as is described in FIG. 4C. Differentanimals can be produced for each host V gene profile. Thus, such ananimal might have only the V genes that are common in a particularfamily or even an individual. Alternatively, an animal might have onlythose V genes that are common for an ethnic group. The advantage is thatthe antibodies can be directly made from such an organism, and thusthere is no concern that one would lose the functionality of theantibody when one was making the gene-optimized antibodies. Of coursegenetically altered mice, methods of making them, as well as their usein creating gene-optimized antibodies are all included as currentembodiments. In one embodiment, the gene-optimized antibody of thetransgenic organism contains no V_(H)3-9, V_(H)3-13, V_(H)3-64 genes, orsome combination thereof. In some embodiments, the transgenic organismcontains no genes that are not present in the population.

In theory, any organism can be used as an antibody producing system. Inone embodiment, all of the low frequency genes and variants thereof areremoved from the organism; thus, when the organism launches an immuneresponse to generate and select antibodies, only antibodies with highfrequency genes will be used. While transgenic organisms can be used,other approaches, such as antisense RNA, siRNA, or antibodies directedtowards protein sections of low frequency genes can also be employed tobias the selection and production of antibodies to those that do notcontain low frequency genes. In one embodiment, a rabbit is the organismof interest, wherein all of the low frequency V genes are removed fromthe rabbit.

In one embodiment, a XenoMouse® mouse is genetically altered to onlyhave high frequency (low-risk) genes; thus, any antibodies produced bythe mouse in response to the antigen will comprise only high frequencygenes. Alternatively, the mouse can be altered to remove all, or someof, of the low frequency genes; thus, any antibody produced will haveno, or fewer, low frequency genes; and thus, have a reduced risk ofinducing a HAHA response when administered to a patient.

As will be appreciated by one of skill in the art, this particulararrangement allows one to produce practically any type of antibody ingeneral, such as catalytic or agonistic (activating), and do so in abackground which is going to result in an antibody with a reducedlikelihood of inducing a HAHA response when administered to a patient.Thus, one does not have to worry about altering the functionality of anantibody when one is trying to optimize it by removing low frequencygenes, since there will be no low frequency genes to begin with.

In one embodiment, several different types of genetically alteredorganisms are created, each missing various low frequency genes orcomprising additional high frequency genes, or both. Each of thedifferent types of genetically altered organisms will have a particularfrequency of occurrence profile. This recognizes the fact that eventhough there are genes that are common throughout the entire populationand genes that are rare throughout the entire population, there may besubsets of the population in which particular genes are common and othergenes are rare, in contrast to the entire population. Thus, it can bedesirable to customize the V gene selection in each genetically alteredorganism for each of these subpopulations, in order to further reducethe likelihood of the antibody inducing a HAHA response. Thus, in oneembodiment, there can be a genetically altered organism comprising onlyV genes that are common in subpopulation 1, while there would be asecond genetically altered organism comprising only V genes that arecommon in subpopulation 2. The result would be a further customizationof the genes used in the antibody creation and thus a further reductionin the risk of the final antibody producing a HAHA response for thatparticular subpopulation. This customization could be useful for anyaspect of the herein-described embodiments.

The subpopulations can be created or individualized in any manner thatthe databases could be organized, which is discussed more fully later.Briefly, in one embodiment, these subpopulations are created based upondatabases that are divided between various ethnic lines. In anotherembodiment, the populations are divided by family medical information.In a further embodiment, the genes selected are based on the patient'sindividual gene set, for instance, their own set of V_(H) genes.

In one embodiment, the genes that are selected from the database are Vgenes. In another embodiment, the genes are V_(H) genes. In anotherembodiment, the genes are V_(H)3 genes. In yet another embodiment, theV_(H)3-9 gene is removed from the selection of possible genes that anorganism may use in the creation and selection of an antibody. Thisresults in an organism that creates antibodies, wherein none of theantibodies have the V_(H)3-9 gene, and thus one has an organism capableof producing antibodies with a reduced risk of inducing a HAHA response.In another embodiment, the gene that is removed is selected from:V_(H)3-9, V_(H)3-13, V_(H)3-64, and some combination thereof.

In some embodiments, the organisms are part of a kit for determining therisk of a HAHA response being induced by a particular antibody. In oneembodiment, the kit comprises a transgenic animal that produces humanantibodies, such as a XenoMouse® mouse, where the animal lacks the sameV genes that the human or population of humans to receive the antibodylack. That is, the V gene set for the animal is the same, orapproximately the same, as the V gene set for the population. In otherwords, the V gene profile of the animal is the same as the V geneprofile of the population. The kit can further comprise an antibody thatwill bind to human antibodies, to detect if a HAHA response hasresulted. Additionally, the kit can comprise an antigenic substance(e.g., TCE), to associate with an antibody to be tested. The kit cancomprise a device for administering an antibody to an animal. In someembodiments, the transgenic animal only has low risk genes. In someembodiments, the transgenic animal only has high risk genes.

Methods of Determining which Modification to Make to Optimize anAntibody to have a Reduced Risk of Inducing a Human Anti-Human Antibody(HAHA) Response.

The genes of an antibody can be modified by various means; FIG. 5 showsseveral simple examples by which the modification can occur. This figureprovides guidance for both how to modify the genes of a potentialorganism, for instance, by removing a risk gene through deletion, and itprovides guidance for how the changes can be made at the protein level,such as through point mutations. In one embodiment, the genes are simplyremoved from the genome, and there is no possibility that the antibodywith the gene will be produced. Thus, gene deletion can achieve thedesired effect. Alternatively, the genetic material may not need to beremoved, rather the expression of the genetic material can be inhibited,such as through siRNA or anti-sense RNA. More preferably, transcriptionof the V gene can be inhibited by altering the noncoding regions of thegenes or by adding specific transcription blockers. Alternatively, theparticular gene can be altered to a sequence which is either more common(thus lower risk), or so that it no longer induces a HAHA response. Forexample, site directed mutagenesis of the gene sequence can besufficient to transform a high-risk gene into a low-risk or neutral riskgene. The benefit of not completely removing the gene is that it allowsgreater V gene diversity.

As will be appreciated by one of skill in the art, the modification of agene can have a substantial impact upon the function of the antibody.Thus, a testing of the functionality and HAHA response induction by theantibody, as described above, can be desirable following eachmodification. Additionally, these tests can allow one to select whichmodification should be made. Standard antibody binding assays will besufficient to determine if any modification has a negative impact on thefunction of the antibody. These assays include surface plasmon resonancetype tests, such as BIAcore affinity measurements and column elutiontype binding assays, appropriate functional assays, as well as manyother techniques known in the art.

Furthermore, similar functional examinations can be achieved for otherfunctionalities of antibodies, such as antigenic antibodies. In suchcases, the appropriate test to determine that the modification of theantibody has not destroyed the usefulness of the antibody can beperformed to determine that the antibody is still useful. Additionally,the in silico tests described above could be used to predict the impactof any gene modification on the paratope of the antibody. Alternatively,in silico tests can be performed to determine if the removal ofparticular genes would be predicted to have detrimental effects on thefunctionality of the protein, such as protein structure modeling,further described herein and in U.S. Pat, Pub. 20040005630 (PublishedJan. 8, 2004, to Studnicka).

As will be appreciated by one of skill in the art, there are a varietyof ways in which these functionalities can be studied, especially withregard to epitope binding by the paratope. One way is to use astructural model generated, perhaps as described herein, and then to usea program such as InsightII (Accelrys, San Diego, Calif.), which has adocking module, which, among other things, is capable of performing aMonte Carlo search on the conformational and orientational spacesbetween the paratope and its epitope. The result is that one is able toestimate where and how the epitope interacts with the paratope. In oneembodiment, only a fragment, or variant, of the epitope is used toassist in determining the relevant interactions. In one embodiment, theentire epitope is used in the modeling of the interaction between theparatope and the epitope. As will be appreciated by one of skill in theart, these two different approaches have different advantages anddisadvantages. For instance, using only a fragment of the epitope allowsfor a more detailed examination of the possible variations of each sidechain, without taking huge amounts of time. On the other hand, by usingonly a fragment of the epitope, or simply the epitope instead of theentire protein, it is possible that the characteristics of the epitopefragment may not be the same as the characteristics for the wholeepitope, thus possibly increasing the risk of being mislead during thecomputational modeling. In one embodiment, both approaches are used to alimited extent, in order to cross check the results. In a preferredembodiment, if a variant of an epitope is used, it will be selected sothat the variant of the epitope comprises the most important residues ofthe epitope. The identity of the most important residues can bedetermined in any number of ways.

With these generated structures, one is able to determine which residuesare the most important in the interaction between the epitope and theparatope. Thus, in one embodiment, one is able to readily select whichresidues to change in order to avoid altering the bindingcharacteristics of the antibody. For instance, it can be apparent fromthe docking models that the side chains of certain residues in theparatope can sterically hinder the binding of the epitope, thus alteringthese residues to residues with smaller side chains can be beneficialfor binding and less likely to induce a HAHA response. One can determinethis in many ways. For example, one can simply look at the two modelsand estimate interactions based on functional groups and proximity.Alternatively, one can perform repeated pairings of epitope andparatope, as described above, in order to obtain more favorable energyinteractions. One can also determine these interactions for a variety ofvariants of the antibody to determine alternative ways in which theantibody can bind to the epitope. One can also combine the variousmodels to determine how one should alter the structure of the antibodiesin order to obtain an antibody with the particular characteristics thatare desired.

For example, consensus sequences for the protein products of the V genescan be created. The V genes can be combined into consensus sequences,either as a function of their structural similarities when translatedinto protein, or through the genes' determined probabilities of inducinga HAHA response. This latter option allows one to develop a model ofwhat a low-risk and what a high-risk protein product from a gene lookslike. Thus, the structure of a protein encoded in part by a V gene,which is responsible for inducing a HAHA response, can be predicted.Additionally, the structures of proteins from a V gene that does notproduce HAHA responses can also be predicted. For instance, in FIG. 2C,it is apparent from the structures that an antibody with gene A willoccur with a frequency of 100%, an antibody with the structure of gene Bwill not occur, and an antibody with the structure of Gene C will alsooccur with a frequency of occurrence of 100%. Additionally, thisinformation can be used to selectively change the genes that areavailable for creating antibodies, as shown in FIG. 6. FIG. 6 displaysthe frequencies of occurrence for three genes, A, B, and C. While onecan observe that gene C has a greater risk of inducing HAHA (in apopulation) than gene A or B, since the protein structures predicted bygenes C and gene B are similar, instead of simply removing gene C froman animal used to produce these proteins, one can substitute a secondcopy of the B gene, or another gene with a structure similar to the Bgene, instead. This replacement by similar structure can also proveuseful in replacing V genes that are high-risk for one population ofpeople, with a structurally similar, but HAHA inducing dissimilar gene.

The structural information can also be used to select particular aminoacids to change in order to reduce the risk of a HAHA response. Oneexample of this is shown in FIG. 7. The amino acid sequences from thegenes are obtained from high-risk and low-risk genes in order to make ahigh-risk consensus sequence and a low-risk consensus sequence. Withboth of these consensus sequences, and then the predicted structure ofan antibody, or V gene thereof, the residues that need to be changedshould be obvious from a comparison of the high risk consensus sequenceto the individual predicted structure. For instance, in FIG. 7, thecomparison between the protein sequence of the V gene and the consensussequence of the high-risk gene, in protein form, suggests that both thefourth and last amino acids are possible targets for modification. Whatthe residues should be changed to will be apparent from a comparison ofthe individual sequence to the low-risk consensus sequence. Forinstance, in FIG. 7, the comparison between the protein sequence of theV gene and the low-risk consensus sequence suggests that the fourthposition should be changed to a noncharged sidechain. Additionally, thissecond comparison suggests that the last position in the A gene is notthat important. It is also important to note that the single amino acidoutside of the section defined as gene A, is not relevant to theanalysis here. It is possible to use only a single consensus sequence;however, this could results in an increased risk of loss offunctionality. One example of a possible alignment of the various genesis shown in FIG. 16A and FIG. 16B, for V_(H) genes.

These consensus sequences and structures therefrom, can be weighted inany manner described herein. For example, if one is given three genes A,1% (high-risk), B 50% (low-risk) and C, 100% (low to no risk) for agiven population, while A and B can be compared to develop a consensussequence, A can have more weight in developing a consensus sequence thatis representative for high risk V genes.

The models determined above can be tested through various techniques.For example, the interaction energy can be determined with the programsdiscussed above in order to determine which of the variants to furtherexamine. In addition, Coulombic and van der Waals interactions are usedto determine the interaction energies of the epitope and the variantparatopes. Also site directed mutagenesis can be used to see ifpredicted changes in antibody structure actually result in the desiredchanges in binding characteristics. Alternatively, changes can be madeto the epitope to verify that the models are correct or to determinegeneral binding themes that can be occurring between the paratope andthe epitope.

The above methods for modeling structures can be used to determine whatchanges in protein structure will result in particular desiredcharacteristics of an antibody. These methods can be used to determinewhat changes in protein structure will not result in the desiredcharacteristics.

As will be appreciated by one of skill in the art, while these modelswill provide the guidance necessary to make the antibodies and variantsthereof of the present embodiments, it may still be necessary to performroutine testing of the in silico models, perhaps through in vitrostudies. In addition, as will be apparent to one of skill in the art,any modification may also have additional side effects on the activityof the antibody. For instance, while any alteration predicted to resultin greater binding may induce greater binding, it may also cause otherstructural changes that might reduce or alter the activity of theantibody. The determination of whether or not this is the case isroutine in the art and can be achieved in many ways.

It is important to realize that a reduction in the functionality of theantibody is not necessarily detrimental to the usefulness of the presentembodiment. Thus, a modification that reduces 99% of the functionalityof the antibody, but also reduces the likelihood of a HAHA responsebeing initiated by as much or more, can still be useful. Indeed, in someembodiments, a greater reduction in functionality than in reducedlikelihood of HAHA response initiation may be sufficient or evendesirable. In one embodiment, the reduction in functionality is one ofthe following: 0% to less than 1%, between 1% and 5%, between 5% and30%, between 30% and 60%, between 60% and 90%, between 90% and 99%.Alternatively, an acceptable decrease in functionality is determined asa function of effective decrease in the risk of a HAHA response beinginduced, either for a population of people, or for a particularindividual. Alternatively, the modification can result in an increase infunctionality. In one embodiment, acceptable decreases in the risk of aHAHA response for any individual in a population is one of thefollowing: less than 1%, between 1% and 5%, between 5% and 30%, between30% and 60%, between 60% and 90%, between 90% and 100%, and 100%.Alternatively, the acceptable decrease can be determined as a functionof the change in functionality, where smaller decreases in the risk of aHAHA response is desirable where little or positive changes occur infunctionality, while larger decreases are desirable where largernegative changes in functionality occur.

By “functionality” it is meant the usefulness of the antibody. Forexample, an antibody may simply be a binding antibody, perhaps useful indetection assays, and requiring tight binding, but not necessarily rapidbinding. Thus, the K_(D) of such an antibody would be the factor thatone would look at to determine if the modification had adverselyimpacted the antibody too much to be useful. Alternatively, if fastbinding was desirable, then the k_(a) would be the primary functionalityexamined following the modification. Alternatively, specificity, eithertowards a particular epitope of an antibody, for instance to allow theantibody to act as a blocker on a receptor, or as regards one targetversus another target, for instance in an in vivo situation to reducethe likelihood of cross reactivity. Alternatively, the antibody may bean agonist type antibody and be useful as a ligand in activatingreceptors. Alternatively, the antibody could be an enzymatic orcatalytic antibody and be functional in the sense that it promotesparticular chemical reactions, such as in U.S. Pat. Nos. 6,043,069,issued to Koentgen et al., Mar. 28, 2000; 5,683,694 issued to Landry,Sep. 7, 1999; and 5,258,289 issued to Davis et al., Nov. 2, 1993). Aswill be appreciated by one of skill in the art, a single antibody mayhave a combination of any of these, as well as many other,functionalities. The relative importance of each can be easilydetermined on a case by case basis.

In one embodiment, the genes compared are V, D, or J genes. In anotherembodiment, the genes are V genes. In another embodiment, the genescompared are V_(H) genes, V_(k), or V_(lambda) genes. In anotherembodiment, the genes are V_(H)3 genes. In one embodiment, the presenceof the V_(H)3-9, V_(H)3-13, and V_(H)3-64 genes, or variants thereof, isan indicator of a high likelihood that the particular antibody will havean increased risk of inducing a HAHA response in some populations. Inanother embodiment, the genes are any of the genes published in Matsudaet al. (J. Exp. Med. 188:2151-2162, (1998)). In one embodiment, it isactually the protein segments that are examined; thus, only functionallycoding V_(H) genes are relevant. In another embodiment, it is only themRNA from the genes that is analyzed or compared. In another embodiment,the segments are only transcribed. In another embodiment, the segmentsare only ORFs. In another embodiment, the segments are only pseudogeneswith point mutations. In another embodiment, the segments are onlypseudogenes with truncations. In one embodiment, the V_(H) gene isselected from one of the following: 1-2, 1-3, 1-8, 1-18, 1-24, 1-45,1-46, 1-58, 1-69, 2-5, 2-26, 2-70, 3-7, 3-9, 3-11, 3-13, 3-15, 3-20,3-21, 3-23, 3-30, 3-33, 3-43, 3-48, 3-49, 3-53, 3-64, 3-66, 3-72, 3-73,3-74, 4-4, 4-31, 4-34, 4-39, 4-59, 4-61, 5-51, 6-1. A more completelisting of V_(H) and other genes can be found in FIG. 8 and FIG. 17Athrough FIG. 22J.

As will be appreciated by one of skill in the art, the minimization ofhigh-risk genes will result in an antibody that has a reduced likelihoodof inducing a HAHA response. However, the optimization of low-risk genescan also be a useful method of achieving the same end goal of anantibody with a lower risk of inducing a HAHA response. Thus, in oneembodiment, the antibodies can be optimized by replacing high-risk geneswith low-risk genes. In one embodiment, this refers to replacing ahigh-risk V gene of an antibody with a structurally similar low riskgene of a V gene.

Methods of Determining the Probability of a HAHA Response Occurring fora Particular Individual for a Given Antibody.

In some situations, the risk of a HAHA response may not be able to beminimized to a desired amount. However, knowing the risk associated witha particular antibody gene set for a particular host V gene profile of apatient can allow people to realize the degree of the risk involved inadministering the particular antibody to the particular patient. Withthis information they can either take preventative measures against aHAHA response, or make the patient fully aware of the risks. Having thisinformation can also aid in the diagnosis of future possible problems,if they occur. These comparison and data analysis techniques are alsogenerally useful for any of the comparisons of high-risk and low-riskgenes.

These calculations can be performed using any of the gene profilesdisclosed herein. This can be done by determining which genes have a lowrisk and high risk of inducing a HAHA response. While knowing theprecise host V gene profile of a patient may be desirable in somesituations, it is also possible to predict the probability that a HAHAresponse will occur by using different types of host V gene profiles,such as those based on ethnic background or broader profiles. One ofskill in the art will appreciate that the ability of these comparisonsto predict such HAHA responses will decrease when less customizedprofiles are used.

In one embodiment, each gene in the particular antibody gene set that isa low-risk gene is given a negative single point value and each genethat is a high risk gene is given a positive value. One can then addeach of the values corresponding to each of the genes in the gene set ofeach antibody to determine the risk of a HAHA response occurring. Themore positive the number, the greater the chance that a HAHA responsewill occur. Thus, if the gene set only involves V genes, the presence ofa high-risk V gene will represent risk. In this embodiment, allhigh-risk V genes will have the same total risk value, namely 1. In someembodiments, only high-risk genes are scored; thus, a gene is given apoint value if it occurs with less than a minimum or predeterminedfrequency of occurrence and a score will indicate that there is a riskof a HAHA response. Higher scores can indicate a greater risk.

In another embodiment that involves a profile of a population, insteadof each risk gene being assigned a single point value, the point valueis determined based upon the frequency of the genes in the profile. Forexample, a gene that occurs with a frequency of 1% is assigned a greaterpoint value than a gene that occurs with a frequency of 10%. Likewise, agene which occurs with a frequency of 99% would be assigned a greaternumber than a gene which occurs with a frequency of 100%. Of course, ifall four genes were being compared, the relative weights of the pointvalue would be 1%>10%>99%>100%. Each of the values for each of the genesare added and the higher the number, the greater the risk of a HAHAresponse occurring. In one embodiment, the point values assigned aredirectly related to their frequency of occurrence in the profile. Forexample, the point value for a gene that has a frequency of occurrenceof 10% in the profile would be approximately 10 fold smaller than thatof a gene, which occurs at 1% in the profile. In the end, the lower thenumber, the lower the risk associated with that antibody. Thus, in theembodiment in which the gene set only contemplates V genes, the totalrisk score or value for the antibody would be the risk value assigned tothat V gene.

Alternatively, one can examine the risk associated with certain genefrequencies in antibodies in inducing a HAHA response when theantibodies are administered to a subject. Thus, in one embodiment,actual previous experimental data is also included in thesecalculations.

In one embodiment, the data can be taken from previous administrationsof antibodies to patients and an examination of whether or not any HAHAresponse was initiated from the use of the antibody. An example of howthis can be done is shown in FIG. 9. This also allows one to correlaterisks in different genetic backgrounds, such as the ethnic backgroundsdiscussed herein. The simple correlation of antibody to HAHA responsewill allow one to determine the probability of an antibody inducing aHAHA response. In one embodiment, illustrated on the left side of FIG.9, one can obtain the degree of risk associated with each V gene byawarding points to the V gene that occurs in an antibody that has beenshown to cause a HAHA response. Each time the antibody is administeredand elicits a HAHA response; the genes of the antibody will be awarded apoint. The genes with the highest point value, or “total risk score” arethe genes most likely to be high-risk genes. In embodiments in which theonly genes examined are the V genes, the point value awarded is onlycorrelated to the risk associated with the V gene.

Alternatively, as shown on the right hand side of FIG. 9, one can applya subtractive process to the analysis. Thus, while those genes thatoccur in antibodies that induce a HAHA response would be awarded a pointvalue, if the same gene appeared in another antibody and did not inducea HAHA response, then one would subtract the points awarded for thisgene. Again, the genes with the highest total risk score will have thegreatest risk of inducing a HAHA response. While such a subtractiveapproach can remove many possible high-risk genes, those genes that areidentified by the process will be high-risk genes. Alternatively, onecan combine the two embodiments to help filter out more of the falsepositives and keep more of the false negatives. This can be done so asto correct for differences in the possible genetic backgrounds of thepatients.

In another embodiment, a humanized organism is used to see if aparticular antibody or gene has a high risk of inducing a HAHA response.This is discussed in greater detail below.

Of course, multiple testings will increase the certainty of whether ornot it is a high-risk antibody. There are at least two methods by whichone can determine if a gene is a high-risk gene for this embodiment.First, after determining a low-risk antibody, which is any antibody thatrarely or, preferably, does not induce a HAHA response, one then altersthe gene set composition of the antibody, substituting in a new V geneand testing the modified antibody. If the modified antibody results in aHAHA response, then the introduced gene could be a high-risk gene.

In another embodiment, a human or XenoMouse® mouse is used as the testsubject to determine if a V gene or an antibody is a high risk compound.

As appreciated by one of skill in the art, the use of these experimentalresults eliminates or greatly reduces the role that a host V geneprofile will play in the current embodiments. However, the correlationof a particular gene with a particular likelihood of a HAHA responsebeing induced is still important.

Such experimental data can be similarly useful in defining high-riskgenes or low-risk genes as well. The above mentioned methods forcalculating the risk of a HAHA response occurring due to a particulargene, gene set, or antibody can be used for the other methods involvingindividual genes as well. Thus, while determining which genes and howthe gene should be modified can involve an analysis of a single gene,the different methods of weighing each gene, discussed above, can beincorporated into their analysis.

In some embodiments, the risk of a HAHA response is calculated for aparticular individual. In one embodiment, the gene set of the candidateantibody to be administered to the individual will be compared to theantibody gene set of the individual. Low risk genes will be those thatthe antibody and the individual have and express and high risk geneswill be those that the antibody has (i.e., encode the protein structureof the antibody) but are not present in the individual's genes.

In another embodiment, the risk of a HAHA response is calculated on abroader basis. For instance, the risk can be calculated from differentnormalized host V gene profiles. Thus, if the host V gene profile is foran entire family, then the risk value produced from the comparison willbe for the entire family. Likewise, if an ethnic host V gene profile isused, then any risk value produced will be for the entire ethnic group.One of skill in the art will recognize that while the accuracy of therisk value will decrease as the variation in the host V gene profileincreases (for example a V gene profile of identical twins will be muchmore accurate than one for an entire ethnic group), there is still avalue in being able to assign a general level of risk to the antibodies.

Any of the additional methods also discussed herein can also be used asdescribed above, in order to correlate a patient's risk of developing aHAHA response following the administration of an antibody. Additionally,even if the antibody being administered is a gene-optimized antibody, itmay still be useful to determine the risk associated with that antibodyfor that patient.

Databases and Methods of Making Host V Gene Profile Databases toDetermine High-Risk and Low-Risk Genes.

At a simple level, one need only define a population for the host V geneprofile and then determine the V genes in that population. The frequencywith which those V genes appear in that population can then bedetermined. In one embodiment, the frequency with which the mRNA appearsfrom the genes is determined. Alternatively, the frequency with whichthe genes are actually used in protein, in the population can bedetermined. Alternatively, either the structure or functionality of theprotein is actually determined. Thus, the mere presence of a V genewould not automatically mean that a V gene was added to the profile tobe counted as a common gene, instead, the protein product would have tobe expressed in the population in order for the gene to be added to theprofile.

As discussed above, these profiles can be directed to the DNA level,mRNA level, amino acid level or protein structure level. The profilescan be directed to the basic building blocks of protein structure or thelarger structure of the antibody. The databases can also factor inaspects of clustering comparing the risk (or probability) associatedwith combinations of particular V_(H) and V_(L) genes within anantibody, or particular V_(H), D_(H) J_(H), and J_(L) genes within anantibody chain. In one embodiment, the databases can include any methodof analyzing genetic material, so long as the defining elements of eachV gene coding region are the primary level at which the analysis occurs.

The profiles can be directed to populations of any size. In oneembodiment, the profiles are split into ethnic groups, as explainedabove. In another embodiment, the profiles are split into family groups.The profile can also be a universal profile as well.

The profiles can be supplemented with additional information fromexperimental findings or test results. For instance, an examination ofthe frequency of V genes in antibodies administered to patients andcorrelating this to those individuals that experienced a HAHA responsecould be very useful in identifying those genes that are so rare thatthey are not included in the profile. An example of this is shown inFIG. 9. This can be a problem, as genes that are very rare not only havethe greatest chance of inducing a HAHA response, but also the leastchance of being in the profile. Fortunately, once a profile is developedthat is sufficient in size; the simple absence of the V gene from theprofile is an indication that the gene is a risk gene. As appreciated byone of skill in the art, there can be some situations where, even thougha V gene is a low-risk gene, its actual use still induces a HAHAresponse. In such circumstances, it is possible for the profile toreflect this fact and adjust the scoring system to predict risk orsuggest gene modification for making gene-optimized antibodiesaccordingly.

As understood by one of skill in the art, many of the adjustments madeto the profile could also be incorporated into the actual comparison ofthe profile to other genes. Such an analysis is acceptable so long asfactors are not inadvertently incorporated into the calculations in aduplicitous manner.

In some embodiments, high risk genes are V_(H)3-9, V_(H)3-13, andV_(H)3-64, for a particular population. As will be appreciated by one ofskill in the art, whether a gene is a high-risk gene or not can dependupon the particular population or individual that is to be host to theantibody encoded by the gene. Thus, it is often helpful to note theparticular population that has the associated risk. As will beappreciated by one of skill in the art, given the present disclosure, acategorization of various genes into high, medium, or low risk, or riskpercentages can be easily achieved, by, for example, following theexamples below.

Gene-Optimized Antibody Proteins, Nucleic Acids, and Variants Thereof, VGene Profile Customized XenoMouse® Mice.

Also included in the present embodiments are the actual gene-optimizedantibodies, the nucleic acids encoding the gene-optimized antibodies andvariants thereof. While the antibodies need not be human, there areadvantages to either human or humanized antibodies.

Human antibodies avoid some of the problems associated with antibodiesthat possess murine or rat variable and/or constant regions. Thepresence of such murine or rat derived proteins can lead to the rapidclearance of the antibodies or can lead to the generation of an immuneresponse against the antibody by a patient. In order to avoid theutilization of murine or rat derived antibodies, fully human antibodiescan be generated through the introduction of human antibody functioninto a rodent so that the rodent produces fully human antibodies.

The ability to clone and reconstruct megabase-sized human loci in YACs(yeast artificial chromosome) and to introduce them into the mousegermline provides a powerful approach to elucidating the functionalcomponents of very large or crudely mapped loci as well as generatinguseful models of human disease. Furthermore, the utilization of suchtechnology for substitution of mouse loci with their human equivalentscould provide unique insights into the expression and regulation ofhuman gene products during development, their communication with othersystems, and their involvement in disease induction and progression.Thus, in one embodiment, the compositions of the embodiments are placedin the mouse germline. This allows one to create an organism with aparticular host gene profile, V gene profile, or normalized V geneprofile which can then be used to make optimized antibodies or V geneoptimized antibodies.

An important practical application of such a strategy is the“humanization” of the mouse humoral immune system. Introduction of humanimmunoglobulin (Ig) loci into mice in which the endogenous Ig genes havebeen inactivated offers the opportunity to study the mechanismsunderlying programmed expression and assembly of antibodies as well astheir role in B-cell development. Furthermore, such a strategy couldprovide an ideal source for production of fully human monoclonalantibodies (mAbs)—an important milestone towards fulfilling the promiseof antibody therapy in human disease. Fully human antibodies areexpected to minimize the immunogenic and allergic responses intrinsic tomurine or murine-derivatized mAbs and thus to increase the efficacy andsafety of the administered antibodies. The use of fully human antibodiescan be expected to provide a substantial advantage in the treatment ofchronic and recurring human diseases, such as inflammation,autoimmunity, and cancer, which require repeated antibodyadministrations.

One approach towards this goal was to engineer mouse strains deficientin mouse antibody production with large fragments of the human Ig lociin anticipation that such mice would produce a large repertoire of humanantibodies in the absence of mouse antibodies. Large human Ig fragmentswould preserve the large variable gene diversity as well as the properregulation of antibody production and expression. By exploiting themouse machinery for antibody diversification and selection and the lackof immunological tolerance to human proteins, the reproduced humanantibody repertoire in these mouse strains should yield high affinityantibodies against any antigen of interest, including human antigens.Using the hybridoma technology, antigen-specific human mAbs with thedesired specificity could be readily produced and selected. This generalstrategy was demonstrated in connection with our generation of the firstXenoMouse® mouse strains as published in 1994. See Green et al. NatureGenetics 7:13-21 (1994). The XenoMouse® strains were engineered withyeast artificial chromosomes (YACs) containing 245 kb and 190 kb-sizedgermline configuration fragments of the human heavy chain locus andkappa light chain locus, respectively, which contained core variable andconstant region sequences. Id. The human Ig containing YACs proved to becompatible with the mouse system for both rearrangement and expressionof antibodies and were capable of substituting for the inactivated mouseIg genes. This was demonstrated by their ability to induce B-celldevelopment, to produce an adult-like human repertoire of fully humanantibodies, and to generate antigen-specific human mAbs. These resultsalso suggested that introduction of larger portions of the human Ig locicontaining greater numbers of V genes, additional regulatory elements,and human Ig constant regions might recapitulate substantially the fullrepertoire that is characteristic of the human humoral response toinfection and immunization. The work of Green et al. was recentlyextended to the introduction of greater than approximately 80% of thehuman antibody repertoire through introduction of megabase sized,germline configuration YAC fragments of the human heavy chain loci andkappa light chain loci, respectively. See Mendez et al. Nature Genetics15:146-156 (1997) and U.S. patent application Ser. No. 08/759,620, filedDec. 3, 1996, the disclosures of which are hereby incorporated byreference.

One method for generating fully human antibodies is through the use ofXENOMOUSE® strains of mice that have been engineered to contain 245 kband 190 kb-sized germline configuration fragments of the human heavychain locus and kappa light chain locus. Other XenoMouse strains of micecontain 980 kb and 800 kb-sized germline configuration fragments of thehuman heavy chain locus and kappa light chain locus. Still otherXenoMouse strains of mice contain 980 kb and 800 kb-sized germlineconfiguration fragments of the human heavy chain locus and kappa lightchain locus plus a 740 kb-sized germline configured complete humanlambda light chain locus. See Mendez et al. Nature Genetics 15:146-156(1997) and Green and Jakobovits J. Exp. Med. 188:483-495 (1998). TheXENOMOUSE® strains are available from Abgenix, Inc. (Fremont, Calif.).

The production of the XenoMouse® mice is further discussed anddelineated in U.S. patent application Ser. Nos. 07/466,008, filed Jan.12, 1990, 07/610,515, filed Nov. 8, 1990, 07/919,297, filed Jul. 24,1992, 07/922,649, filed Jul. 30, 1992, filed 08/031,801, filed Mar. 15,1993, 08/112,848, filed Aug. 27, 1993, 08/234,145, filed Apr. 28, 1994,08/376,279, filed Jan. 20, 1995, 08/430, 938, Apr. 27, 1995, 08/464,584,filed Jun. 5, 1995, 08/464,582, filed Jun. 5, 1995, 08/463,191, filedJun. 5, 1995, 08/462,837, filed Jun. 5, 1995, 08/486,853, filed Jun. 5,1995, 08/486,857, filed Jun. 5, 1995, 08/486,859, filed Jun. 5, 1995,08/462,513, filed Jun. 5, 1995, 08/724,752, filed Oct. 2, 1996, and08/759,620, filed Dec. 3, 1996 and U.S. Pat. Nos. 6,162,963, 6,150,584,6,114,598, 6,075,181, and 5,939,598 and Japanese Patent Nos. 3 068 180B2, 3 068 506 B2, and 3 068 507 B2. See also Mendez et al. NatureGenetics 15:146-156 (1997) and Green and Jakobovits J. Exp. Med.188:483-495 (1998). See also European Patent No., EP 0 463 151 B1, grantpublished Jun. 12, 1996, International Patent Application No., WO94/02602, published Feb. 3, 1994, International Patent Application No.,WO 96/34096, published Oct. 31, 1996, WO 98/24893, published Jun. 11,1998, WO 00/76310, published Dec. 21, 2000, WO 03/47336. The disclosuresof each of the above-cited patents, applications, and references arehereby incorporated by reference in their entirety.

In an alternative approach, others, including GenPharm International,Inc., have utilized a “minilocus” approach. In the minilocus approach,an exogenous Ig locus is mimicked through the inclusion of pieces(individual genes) from the Ig locus. Thus, one or more V_(H) genes, oneor more D_(H) genes, one or more J_(H) genes, a mu constant region, anda second constant region (preferably a gamma constant region) are formedinto a construct for insertion into an animal. This approach isdescribed in U.S. Pat. No. 5,545,807 to Surani et al. and U.S. Pat. Nos.5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429,5,789,650, 5,814,318, 5,877,397, 5,874,299, and 6,255,458 each toLonberg and Kay, U.S. Pat. Nos. 5,591,669 and 6,023.010 to Krimpenfortand Berns, U.S. Pat. Nos. 5,612,205, 5,721,367, and 5,789,215 to Bernset al., and U.S. Pat. No. 5,643,763 to Choi and Dunn, and GenPharmInternational U.S. patent application Ser. No. 07/574,748, filed Aug.29, 1990, 07/575,962, filed Aug. 31, 1990, 07/810,279, filed Dec. 17,1991, 07/853,408, filed Mar. 18, 1992, 07/904,068, filed Jun. 23, 1992,07/990,860, filed Dec. 16, 1992, 08/053,131, filed Apr. 26, 1993,08/096,762, filed Jul. 22, 1993, 08/155,301, filed Nov. 18, 1993,08/161,739, filed Dec. 3, 1993, 08/165,699, filed Dec. 10, 1993,08/209,741, filed Mar. 9, 1994, the disclosures of which are herebyincorporated by reference. See also European Patent No. 0 546 073 B1,International Patent Application Nos. WO 92/03918, WO 92/22645, WO92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO96/14436, WO 97/13852, and WO 98/24884 and U.S. Pat. No. 5,981,175, thedisclosures of which are hereby incorporated by reference in theirentirety. See further Taylor et al., 1992, Chen et al., 1993, Tuaillonet al., 1993, Choi et al., 1993, Lonberg et al., (1994), Taylor et al.,(1994), and Tuaillon et al., (1995), Fishwild et al., (1996), thedisclosures of which are hereby incorporated by reference in theirentirety.

Kirin has also demonstrated the generation of human antibodies from micein which, through microcell fusion, large pieces of chromosomes, orentire chromosomes, have been introduced. See European PatentApplication Nos. 773 288 and 843 961, the disclosures of which arehereby incorporated by reference.

Xenerex Biosciences is developing a technology for the potentialgeneration of human antibodies. In this technology, SCID mice arereconstituted with human lymphatic cells, e.g., B and/or T cells. Miceare then immunized with an antigen and can generate an immune responseagainst the antigen. See U.S. Pat. Nos. 5,476,996, 5,698,767, and5,958,765.

Human antibodies can also be derived by in vitro methods. Suitableexamples include, but are not limited to, phage display (CAT, Morphosys,Dyax, Biosite/Medarex, Xoma, Symphogen, Alexion (formerly Proliferon),Affimed) ribosome display (CAT), yeast display, and the like.

Antibodies

As discussed above, there are substantial benefits of making transgenicsthat contain only a subset of the possible V genes, in particular, asubset of V_(H) or V_(kappa) or V lambda genes. Thus, a transgenicXenoMouse® animal that lacks certain high-risk genes is particularlydesirable for producing antibodies. However, as appreciated by one ofskill in the art, there is no need that the transgenic has to be a mouseor a XenoMouse® mouse.

Antibodies, as described herein, can be prepared through the utilizationof the XenoMouse® technology, as described below. Such mice, then, arecapable of producing human immunoglobulin molecules and antibodies andare deficient in the production of murine immunoglobulin molecules andantibodies. Technologies utilized for achieving the same are disclosedin the patents, applications, and references cited herein. Inparticular, however, a preferred embodiment of transgenic production ofmice and antibodies therefrom is disclosed in U.S. patent applicationSer. No. 08/759,620, filed Dec. 3, 1996 and International PatentApplication Nos. WO 98/24893, published Jun. 11, 1998 and WO 00/76310,published Dec. 21, 2000, the disclosures of which are herebyincorporated by reference. See also Mendez et al. Nature Genetics15:146-156 (1997), the disclosure of which is hereby incorporated byreference.

Through use of such technology, fully human monoclonal antibodies to avariety of antigens can be produced. Essentially, XenoMouse® lines ofmice are immunized with an antigen of interest, lymphatic cells arerecovered (such as B-cells) from the mice that expressed antibodies, andsuch cells are fused with a myeloid-type cell line to prepare immortalhybridoma cell lines, and such hybridoma cell lines are screened andselected to identify hybridoma cell lines that produce antibodiesspecific to the antigen of interest. Antibodies produced by such celllines are further characterized, including nucleotide and amino acidsequences of the heavy and light chains of such antibodies.

Alternatively, instead of being fused to myeloma cells to generatehybridomas, the antibody produced by recovered cells, isolated fromimmunized XenoMouse® lines of mice, are screened further for reactivityagainst the initial antigen. Such screening includes ELISA, in vitrobinding to cells stably expressing the antigen. The antibodies can alsobe screened to determine if it has a risk of inducing a HAHA response.Single B cells secreting antibodies of interest are then isolated usingan antigen-specific hemolytic plaque assay (Babcook et al., Proc. Natl.Acad. Sci. USA, i93:7843-7848 (1996)). Cells targeted for lysis arepreferably sheep red blood cells (SRBCs) coated with the antigen. In thepresence of a B cell culture secreting the immunoglobulin of interestand complement, the formation of a plaque indicates specificantigen-mediated lysis of the target cells. The single antigen-specificplasma cell in the center of the plaque can be isolated and the geneticinformation that encodes the specificity of the antibody is isolatedfrom the single plasma cell. Using reverse transcriptase PCR, the DNAencoding the variable region of the antibody secreted can be cloned.Such cloned DNA can then be further inserted into a suitable expressionvector, preferably a vector cassette such as a pcDNA, more preferablysuch a pcDNA vector containing the constant domains of immunoglobulinheavy and light chain. The generated vector can then be transfected intohost cells, preferably CHO cells, and cultured in conventional nutrientmedia modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

B cells from XenoMouse® mice can be also be used as a source of geneticmaterial from which antibody display libraries can be generated. Suchlibraries can be made in bacteriophage, yeast or in vitro via ribosomedisplay using ordinary skills in the art. Hyperimmunized XenoMouse® canmay be a rich source from which high-affinity, antigen-reactiveantibodies may be isolated. Accordingly, XenoMouse® mice hyperimmunizedagainst an antigen can be used to generate antibody display librariesfrom which high-affinity antibodies against an antigen may be isolated.Such libraries could be screened against an appropriate target such asan oligopeptide or protein and the resultingly derived antibodiesscreening against cells expressing an antigen to confirm specificity forthe natively display antigen. Full IgG antibody may then be expressedusing recombinant DNA technology. See e.g., WO 99/53049.

In general, antibodies produced by the above-mentioned cell linespossess fully human IgG heavy chains with human light chains. Theantibodies possess high affinities, typically possessing K_(d)'s of fromabout 10⁻⁹ through about 10⁻¹³ M, when measured by either solid phaseand solution phase.

As will be appreciated, antibodies as described herein can be expressedin cell lines other than hybridoma cell lines. Sequences encodingparticular antibodies can be used for transformation of a suitablemammalian host cell. Transformation can be by any known method forintroducing polynucleotides into a host cell, including, for examplepackaging the polynucleotide in a virus (or into a viral vector) andtransducing a host cell with the virus (or vector) or by transfectionprocedures known in the art, as exemplified by U.S. Pat. Nos. 4,399,216,4,912,040, 4,740,461, and 4,959,455 (which patents are herebyincorporated herein by reference). The transformation procedure useddepends upon the host to be transformed. Methods for introduction ofheterologous polynucleotides into mammalian cells are well known in theart and include dextran-mediated transfection, calcium phosphateprecipitation, polybrene mediated transfection, protoplast fusion,electroporation, encapsulation of the polynucleotide(s) in liposomes,and direct microinjection of the DNA into nuclei.

Mammalian cell lines available as hosts for expression are well known inthe art and include many immortalized cell lines available from theAmerican Type Culture Collection (ATCC), including but not limited toChinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK)cells, monkey kidney cells (COS), human hepatocellular carcinoma cells(e.g., Hep G2), and a number of other cell lines.

The XenoMouse® Animal as an Assay System for Determining the Risk ofHAHA Response Induction

Types of XenoMouse® Animals

In one embodiment, a humanized organism is used to see if a particularantibody or gene encoding for part of an antibody has a risk of inducinga HAHA response. In one embodiment, the organism is a XenoMouse® animal.Thus, if an antibody administered to a XenoMouse® animal results in aHAHA response in the animal, then the antibody will be a high-riskantibody for any patient with a immunogenic gene set that is similar tothe XenoMouse® animal's immunogenic gene set. In particular, the similarabsence of high-risk genes between a patient and the XenoMouse® animalwill determine if the XenoMouse® animal is an appropriate model for thatpatient.

In one embodiment, the XenoMouse® animal is customized for an individualor for a group of individuals, such as an ethnic group as discussedabove. For example, a customized XenoMouse® animal will lack the samehigh-risk genes that the patient, for whom the XenoMouse® animal iscustomized, lacks. Thus, a patient of a particular ethnic background canuse a customized XenoMouse® animal that lacks the same high-risk genesthat the patient and the ethnic group lack as a test subject todetermine if a particular antibody will result in a HAHA response in thepatient. Thus, the risk of that patient experiencing a HAHA response ismore accurately assessed. This also allows antibody administration topatients to be more customized.

In one embodiment, the XenoMouse® animal only has the same low riskgenes that the patient or population, which is to accept the antibody,has. Other customized XenoMouse® animal test subjects can also becreated once a trend of high risk or low risk genes is identified forany group of people. A risk profile can be turned into a XenoMouse®animal that is an indicator of risk that a HAHA response will occur ifthe Ab is administered. In one embodiment, the XenoMouse® animal has aminority of the high-risk genes for a population it is to represent. Inanother embodiment, the XenoMouse® animal has from about 50% or fewer ofthe high-risk genes for a population to be treated, for example 50-20,20-10, 10-5, 5-1, or 1-0%. In another embodiment, the XenoMouse® animalhas none of the high-risk genes for a population to be treated with anantibody. In another embodiment, the XenoMouse® animal only has low riskgenes from a consensus of a population of people. In one embodiment, theconsensus is across ethnic groups. In one embodiment, the consensus iswithin ethnic groups.

In one embodiment, the XenoMouse® animal lacks all high risk genes andmedium risk genes. In one embodiment, the high risk genes includeV_(H)3-9, V_(H)3-13, and V_(H)3-64. In one embodiment, low risk geneswill be those that are not high-risk genes. In some embodiments, theXenoMouse® animal only contains low risk genes.

In one embodiment, the XenoMouse® animal comprises all, or substantiallyall, of the V, D, and/or J genes from Homo sapiens. Such a XenoMouse®animal should not display a HAHA response to human antibodies and can beuseful as a negative control to demonstrate an absence of a HAHAresponse.

Methods of Using a XenoMouse® Animal for Detection or Determination ofthe Risk of Inducing a HAHA Response

In one embodiment, a method of using a XenoMouse® animal to determine ifan antibody will induce a HAHA response in a human is provided. Themethod involves administering a candidate antibody to a XenoMouse®animal, allowing a sufficient amount of time to pass so that a HAHAresponse can occur, withdrawing a sample from the XenoMouse® animal,testing the sample for the presence of antibodies directed against thetest antibody, and examining the sample for the presence or absence ofsubstances (e.g., antibodies) that bind to the test antibody. An Exampleof such a use is shown in Example 7 below.

In one embodiment, two antibodies are given to two similar XenoMouse®animals. One antibody will be a candidate antibody, whose HAHA inducingactivity is uncertain, the second antibody will be a control antibodywhose induced HAHA response will be used as a reference point againstthe test antibody's HAHA response. Any antibody with a known orpredicted risk for inducing a HAHA response can be used as a control.

For example, one possible control antibody is HUMIRA®, antibody D2E7,which is known to induce a HAHA response in humans (SEQ ID NO.: 1-4,LCVR, HCVR, LC CDR3, HC CDR3, respectively, FIG. 23A). The resultingHAHA response from the administration of such an antibody to aXenoMouse® animal can be seen in FIG. 12A through FIG. 12C. It isevident that the administration of D2E7 to the XenoMouse® animalsresulted in a significant amount of immunogenicity. On the other hand, acandidate human IgG1, kappa antibody, A, did not induce a significantHAHA response (See, FIGS. 11A-C). The candidate antibody, antibody A, isan antibody described in co-pending U.S. Pat. Application 60/430,729,filed Dec. 2, 2002, herein incorporated in its entirety by reference andhas a sequence identified in SEQ ID NO.: 71-74 in that application, andSEQ ID NO.: 5-8 in the present specification (FIG. 23A).

Any XenoMouse® animal, or other transgenic organism, that has theability to generate human or humanized antibodies can be used. In oneembodiment, a XenoMouse® animal that has no high-risk genes of a humancan be used. Such “universal” organisms are useful in that they have thegreatest ability to detect if there is a risk of the induction of a HAHAresponse. Such a XenoMouse® animal may not be as useful as anotherXenoMouse® animal with a different gene set in predicting if aparticular antibody will induce a HAHA response in a particular patient,as such a XenoMouse® animal may result in many false positives asconcerns individual members of a population. However, in one embodiment,universal mice are useful in identifying universal antibodies that willnot induce a HAHA response across a population of people. For example,while a transgenic organism that lacked all high-risk genes wouldexhibit a HAHA response to any antibody with a high risk gene for anyonein a population, any antibody that did not induce a HAHA response willhave a low probability of inducing a HAHA response in anyone in thepopulation. Thus, such organisms are useful in screens for universalHAHA antibodies.

In one embodiment, high-risk genes that are not in the patient's geneset are not in the customized XenoMouse® animal to which the testantibody will be administered. By using these customized XenoMouse®animals, one is able to reduce the number of false positives that mayoccur. In one embodiment, the customized XenoMouse® animal and thepatient lack all of the same high-risk genes. In another embodiment, thecustomized XenoMouse® animal and the patient or the population that thepatient belongs to lack 100% or fewer of the same high-risk genes, forexample 100-99, 99-98, 98-95, 95-90, 90-80, 80-70, 70-50, 50-30, 30-1,1-0 percent of the same high-risk genes. In another embodiment, thecustomized XenoMouse® animal has the same gene set for V, D, and J genesas the patient or population that the patient belongs to. In oneembodiment, the customized XenoMouse® animal and the patient orpopulation have at least 1 gene in common. For example, they may have100, 100-90, 90-70, 70-50, 50-30, 30-10, 10-1, or 1-0 percent of thesame V, D, or J genes in common.

In another embodiment, the XenoMouse® animal need not be customizedexactly to the patient that is going to receive the antibody. Thus, theXenoMouse® animal can have a high-risk gene that the patient lacks;thus, raising the possibility that the administration of an antibody tothe XenoMouse® animal will result in a false negative. However, theadministration of the antibodies to the XenoMouse® animal can take thisinto account. Antibodies with that particular high-risk gene will not beincluded as possible antibodies for the patient, even if no HAHAresponse is created by the XenoMouse® animal.

Any method of administration that allows a HAHA response to occur or bemonitored is adequate for the purposes of these embodiments. In oneembodiment, the test or candidate antibody is administered into the miceusing an approach similar to what would be used in patients. In anotherembodiment, methods that optimize the likelihood of inducing a HAHAresponse can be useful. For example, as shown in FIGS. 12A-C, whileintravenous administration can lead to the detection of a HAHA responsein the mouse of an antibody that can induce a HAHA response, higherresponses are produced when the antibody is given subcutaneously or withan adjuvant. In one embodiment, the antibody is administered as aprotein or as a nucleic acid.

Additionally, as HAHA responses are more likely to occur after multipleadministrations of an antibody, it can be advantageous to repeatedlyadminister the test antibody to the XenoMouse® animal to see if a HAHAresponse will be induced.

The immunogenic response can be measured by any method, as long as itreveals the presence of antibodies directed to the original testantibody. For example, as discussed in Example 7, a bridging ELISA assaycan be used. Alternatively, as the original test antibody is generallyknown, it is easy to apply the test antibody to beads or on a chip, suchas a BIAcore assay system, to detect if there are proteins in the testsubject's serum sample that bind to the candidate or test antibody.Binding of a substance to the test antibody indicates the presence of apossible immunogenic response. In one embodiment, the particularresponse examined for is a HAHA response. This can be done byadministering a human antibody, or humanized antibody, to a XenoMouse®animal.

In general, the level of immunogenic response generated by an antibodycan be compared to either a positive or negative control antibody. Inone embodiment, whether or not an immunogenic or HAHA response isgenerated is a binary answer, with any deviation from the negativecontrol or set standard indicating that a HAHA response has occurred. Inanother embodiment, the presence or absence of a HAHA response iscorrelated with time or concentration of the antibody administered. Thepresence or absence of a response can also factor in the method ofadministration. In one embodiment, a positive HAHA response is aresponse that displays an amount of a substance that binds to theantibody greater than the amount in a negative control, or a setnegative response standard, in an amount of at least above 100 percentof a negative control, for example, 101, 101-200, 200-300, 300-500,500-800, 800-1500, 1500 percent or more, of the negative control. In oneembodiment, the substance that binds to the administered antibody isdefined as a host antibody; thus, only an increase in host antibodiesthat bind to the administered, or test, antibody will be encompassed bythe term “substance that binds to the administered antibody.” In anotherembodiment, there need be no increase in a substance that binds to theadministered antibody. For example, the host may already have anestablished immunogenic response to the administered antibody. Thus, anysubstantial amount of binding to the administered antibody, even if itdoes not later result in an increase in a substance that binds to theadministered antibody, can indicate that the antibody will induce a HAHAresponse. Such comparisons of data are usually made through an expectedor standard of responses.

The significance of the response will depend upon many factors, such ashow long the response takes to occur in addition to factors such as theneed and benefit of the antibody compared to the risk of a HAHA responseoccurring and possible medication that may be co-administered with theantibody.

One benefit of knowing the risk of a HAHA response being induced iswhether or not other agents should be added with the antibody to reducethe risk of a HAHA response occurring.

One additional use of these XenoMouse® animals and the disclosed methodsis that the XenoMouse® animals and the HAHA inducing antibodies, can becombined to screen for drugs or substances that are able to decrease therisk of a HAHA response occurring (anti-HAHA response, or HAHA inhibitorcompounds). For example, given an antibody that induces a HAHA responsein a XenoMouse® animal, a candidate anti-HAHA response compound can beadded to the XenoMouse® animal, in addition to the HAHA inducingantibody, in order to determine if the candidate anti-HAHA responsecompound is adequate to reduce the risk of a HAHA response occurring.Similarly, XenoMouse® animal with antibodies that induce a HAHA responsecan be used to screen for substances that block a HAHA response (HAHAinducing antibodies). Similarly, the mice and antibodies can be used tohelp determine the effective dosage of the compounds.

In some embodiments, the above substances and methods of administeringthe antibodies are varied so that other factors associated with theinduction of a HAHA response can be examined. As discussed in moredetail below, the particular route of administration, or frequency ofadministration can be altered, while keeping the particular antibody thesame, to determine whether the altered condition affects the HAHAresponse. Additionally, substances other than antibodies can beadministered to determine if the alternative substances inhibit orexacerbate a HAHA response.

As appreciated by one of skill in the art, the above discussionoccasionally details the XenoMouse® animal and methods of its use withregard to high-risk V genes; however, the genes may also be D or J genesas well. Additionally, both light and heavy chain genes are considered.

Eliminating or Minimizing Additional Variables in the Above Methods

As appreciated by one of skill in the art, there are additional possibledifferences between the mouse and human systems that could complicatethe application of any transgenic organism as a detector of HAHAinduction risk. One such factor, discussed below, is the role andpossible differences in the T cell epitope repertoire. This factor andthe theories behind it are for means of illustration only and are notintended as limitations upon the present invention.

B cell-mediated antibody responses to protein antigens are primarilydependent on T cell help in the form of cognate interactions and solublefactors released by activated T cells. This help is triggered by theactivation of T-helper (Th) cells upon recognition of the antigen onceit is has been taken up and proteolytically processed by antigenpresenting cells (APC). Such recognition occurs when the T cell receptor(TCR) on a Th cell binds specifically to the combination of anantigen-derived peptide presented in the groove of majorhistocompatibility complex (MHC) class II heterodimer molecules on thesurface of the APC. Peptide recognition by a Th cell is dependent onboth the ability of the class II MHC to bind the peptide (which isgenetically constrained as it is dependent on the haplotype of the classII MHC molecule), and the ability of the TCR to recognize that peptidesequence (“T cell epitope” or TCE) in the context of class II MHC.

When immunization with a protein antigen fails to elicit a B cellresponse (observed as antibodies produced by such B cells and present inthe serum), several causes may exist. For example, it may reflecttolerance mechanisms at work in the organism, whether T cell or B celltolerance, that lead to an inability to activate the immune response atsome level. Alternatively, it is recognized that proteolytic antigenprocessing, as well as class II MHC presentation constraints, result ina finite repertoire of antigen-derived peptides being presented to Thcells. Consequently, it is possible that a particular antigen may yieldpeptides that either are not presented by class II MHC molecules of theorganism's expressed haplotype, or do not represent T cell epitopes thatcan be recognized by TCR on Th cells. Collectively, such a peptiderepertoire fails to elicit Th cell activation and consequently theevents leading to B cell activation and antibody production do notoccur. When using nonhuman organisms to predict immunogenicity inhumans, the caveat exists that the T cell epitope repertoire in suchorganisms may differ from those present in humans, owing to thedifference in class II MHC and the peptides that they can present. Thiscould result in “false negative” results, in which the presence of Bcell epitopes on an antigen (that is, epitopes recognized by antibodiesproduced by a B cell) may not accurately be reported.

There are several ways to address this possibility; one method isfurther discussed in Example 8 below. Broadly characterized, one canassociate an antigenic substance with the antibody to be tested. For thepurposes of this discussion, an antigenic substance is one that iscapable of inducing an antigenic response. For example, it is capable ofstimulating the production of an antibody. In one embodiment, thesubstance is antigenic in mice, humans, or mammals in general. Inanother embodiment, the substance is antigenic in the organism to whichit is administered.

For example, a humanized IgG1,κ can be coupled to an antigenic proteinsequence, for example, a synthetic peptide having the sequenceCQYIKANSKFIGITELKK (SEQ ID NO: 9) (herein referred to as “T CellEpitope” or “TCE” This sequence has been described as being universallyantigenic (Panina-Bordignon, P., Tan, A., Termijtelen, A., Demotz, S.,Corradin, G. P., and Lanzavecchia, A., Eur. J. Immunol. 19, 2237-2242(1989)). When the combined antibody and antigenic substance areadministered to the organism in which it is to elicit an immunogenicresponse, the presence of the antigenic substance will reduce the riskof the false negatives discussed above, as the antigenic substance willavoid the possible T cell receptor and/or major histocompatibilitycomplex compatibility issues. Even in the absence of endogenous T cellepitopes the test antibody will elicit an antibody response if itpossesses endogenous B cell epitopes (antibody binding sites). In oneembodiment, a “TCE” is any exogenous peptide conjugated to a testarticle.

The antigenic segment can be attached to the antibody in any number ofways, as long as the antigenic segment can still function appropriatelyand as long as it does not unduly prevent the processing and recognitionof the test antibody. In one embodiment, maleimide chemistry can be usedto connect the antigenic segment to the test antibody. For example, asulfo-SMCC can be used. It contains an NHS ester that reacts with amineson the antibody, and a maleimide group that reacts with the sulfhydrylgroup on the N-terminal cysteine of the TCE peptide to generateTCE-decorated antibody (“Ab-TCE”). As controls, additional antibodiescan be treated identically, but without addition of TCE to the reaction(“Ab-sham”). These, along with the untreated antibody and TCE alone canthen be administered to XenoMouse® animals. The results of such anexperiment are described in detail below in connection with Example 9.As will described in detail below, the only samples that resulted in asignificant induction of antibodies directed against the administeredantibody was the antibody conjugated to TCE and the antibodyadministered via base of tail followed by intraperitoneally, in thepresence of adjuvant. Thus, it appears that the use of TCE caneffectively increase the probability of allowing the antibody to inducea HAHA response in a XenoMouse® animal. One can therefore use theXenoMouse® animal to effectively test for factors that increase the riskof a HAHA response occurring.

Transgenic XenoMouse® Animals for Testing for HAHA Response Inhibitors.

In one embodiment, a XenoMouse® animal that exhibits a HAHA response toan antibody is used to screen for possible anti-HAHA compounds (AHAHAcompounds or HAHA inhibitor compounds). For example, given that theXenoMouse® animal will exhibit a HAHA response when it is administered aHAHA inducing antibody, any substance that is administered to theXenoMouse® animal that blocks the induction of the expected HAHAresponse when the HAHA inducing antibody is administered to theXenoMouse® animal will be useful in preventing the induction of a HAHAresponse in a human system as well. Thus, in one embodiment, aXenoMouse® animal that exhibits a HAHA response with respect to aparticular antibody is contemplated as a means for testing variouscompounds for the compounds' ability to inhibit a HAHA response. Theresulting compounds can be effective for preventing the induction ofHAHA responses generally, or for preventing the induction of a HAHAresponse for the particular antibody responsible for inducing the HAHAresponse.

In another embodiment, the compound to be tested for inhibiting HAHAresponses, a candidate HAHA inhibitor, is administered to the XenoMouse®animal before, during, or after the HAHA inducing antibody isadministered to the XenoMouse® animal. The XenoMouse® animal may alsoproduce the HAHA inducing antibody internally as well. For example, theXenoMouse® animal may also be a transgenic mouse for the HAHA inducingantibody protein in question. Alternatively, the XenoMouse® animals canhave a limited number of cells that are producing the protein.Alternatively, the XenoMouse® animal can produce the antibodies throughthe use of viruses or viral constructs to infect the cells of theXenoMouse® animal. Unless otherwise required, the HAHA inducing antibodycan be administered to the XenoMouse® animal test subject in any mannerthat allows a HAHA response.

In one embodiment, a method for testing for HAHA inhibitors involvesadministering both 1) one or more candidate HAHA inhibitors and 2) aHAHA inducing antibody to a XenoMouse® animal. If necessary a continuoussupply of either or both of the candidate HAHA inhibitor and the HAHAinducing antibody will be administered to the XenoMouse® animal for aslong as would have been required for the onset of a HAHA response ifthere had been no HAHA inhibitor is present. A sample may then be takenfrom the XenoMouse® animal to determine if a HAHA response occurred. Ifno, or a lesser amount of a HAHA response occurred, then the candidateHAHA inhibitor will be considered a HAHA inhibitor. A HAHA inhibitorneed not block 100% of a HAHA response. For example, the HAHA inhibitorwill block at least some of the HAHA response, for example, 1, 2, 3-5,5-7, 7-10, 15-20, 20-30, 30-50, 50-70, 70-80, 90-95, 95-97, 98, 99, or100 percent of the HAHA response. This response can be measured indifferent ways, for example, by looking for substances that bind to theHAHA inducing antibody in the XenoMouse® animal's serum. A functionalHAHA inhibitor will reduce or eliminate the presence of antibodiesdirected to the HAHA inducing antibody under a given set of conditions.In one embodiment, the HAHA inhibitor will delay the onset of a HAHAresponse. Thus, a HAHA response and the size of the HAHA response willstill occur, it will simply take longer for that response to occur. TheHAHA inhibitor will delay the response by at least 1 percent, forexample, 1-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80,80-90, 90-100, 100-150, 150-200, 200-300 percent or more.

Optionally, an additional screening step can be performed to makecertain that the HAHA inducing Ab is still functional for its bindingability in the presence of the HAHA inhibitor. Of course, as the HAHAinhibitor is also being administered to a XenoMouse® animal, immunogenicresponses to the inhibitor can also be examined.

A HAHA inducing antibody is an antibody that induces a HAHA responsewhen administered to a patient. This can be determined experimentally,as shown in FIGS. 11A-D and 12A-D. This can also be determined byexamining the frequency of the genes and which genes are high risk andlow risk genes. Antibodies comprising low frequency genes will be HAHAinducing antibodies for those people for which the genes are lowfrequency genes (e.g., absent from an individual).

While XenoMouse® animals have been used as an example, other organisms,with similar human based immune systems will also work for the describedcompositions and methods.

In some embodiments, the various conditions involved in a particulartreatment or experiment can be tested with the use of a XenoMouse® mouseand an antibody. “Condition” is meant to encompass any aspect of thetreatment or experiment that can be altered and includes, for example,altering various steps in the method of treatment or altering theparticular substances being administered. A “first condition” and a“second condition” describe a same aspect of the method or material thatis varied between experiments in order to determine the impact of thecondition on the experiment. In other words, a condition can be theexpression system. Thus, a first condition can be using expressionsystem “X”, while a second condition can be using expression system “Y.”Alternatively, a condition can be the amount of antibody administeredand a first condition can be “X” micrograms while the second conditioncan be “Y” micrograms. Thus, a first condition and a second conditionare alternatives for a particular step or composition.

The importance of the condition in general, as well as a comparison of afirst condition vs. a second condition, can be tested with the abovemice, or other similar transgenic organisms. Examples of conditions arediscussed in the following paragraphs, and include, for example,nonvariable regions of the antibody, the amount of antibodyadministered, the route of administration, the frequency ofadministration and the compositions that are administered together withthe antibody. Thus, methods or compositions with a minimal or reducedrisk of inducing a HAHA response can be identified by altering eachcondition and determining the effect of the altered condition on theHAHA response.

As will be appreciated by one of skill in the art, there are numerousmethods by which different conditions can be tested for their ability toinduce or suppress a HAHA response. In one embodiment, the impact that aparticular condition has on the induction of a HAHA response isdetermined by 1) determining an immunogenic response in a first methodor process using a first condition, 2) determining a second immunogenicresponse in a second method or process using a second condition, and 3)comparing the two responses.

In a preferred embodiment, other steps or compositions in the experimentare maintained while only a single condition is changed. For example,the method can involve 1) providing a first transgenic mouse that has ahuman gene configured to allow the first transgenic mouse to produce afully human or humanized antibody, 2) administering to the firsttransgenic mouse a first foreign antibody under a first condition, 3)determining a presence of a HAHA response in the first transgenic mouse,4) providing a second transgenic mouse that comprises a human geneconfigured to allow the second transgenic mouse to produce the fullyhuman or humanized antibody, 5) administering to the second transgenicmouse a second foreign antibody under a second condition, wherein saidfirst condition and said second condition are variations of the samevariable, 6) determining a presence of a HAHA response in the secondtransgenic mouse, and 7) comparing the presence of the HAHA response inthe first transgenic mouse to the presence of the HAHA response in thesecond transgenic mouse, thereby determining which condition results ina greater HAHA response.

As will be appreciated by one of skill in the art, various subsets ofthe above methods can also be performed, for example, steps 1-3 only. Aswill be appreciated by one of skill in the art “foreign antibody” merelymeans that the antibody is encoded by a gene that is not expressed inthe animal host. “Foreign antibody” can include vectors or constructsencoding for the protein as well.

In some embodiments, the first transgenic mouse and the secondtransgenic mouse are different mice but produce a same fully human orhumanized antibody. For example, the mice are genetically identical intheir ability to produce a fully human antibody. In some embodiments,the first foreign antibody and the second foreign antibody are the sameantibody or have the same structure. This is not meant to denote thatthe same foreign antibody molecule is used in step 2 and 5, but that theforeign antibodies are the same on the protein or nucleic acid level.Thus, the foreign antibodies can be the same and the mice can be thesame type of mice. In a preferred embodiment, the only substantivedifference between steps 1-3 and 4-6 is the first and second condition.As will be appreciated by one of skill in the art, the first and secondforeign antibody and the first and second transgenic mouse can also bereferred to as “foreign antibody” or “transgenic mouse” when the same orsame type of antibody or mouse is being used.

An example of the general method described above is shown in Example 7below. The example demonstrates how the level of immunogenicity dependsupon the condition of route of administration of the antibody and howthis can be determined for mice via S.C. administration (a firstcondition) or via I.V. administration (a second condition). As shown inthe results, S.C. administration resulted in 5:7 mice exhibitingimmunogenicity, while I.V. administration resulted in only 3:7 miceexhibiting immunogenicity. Thus, I.V. administration, for thisparticular experiment, has a lower chance of inducing a HAHA response.

In some embodiments, the effect of antibody isotype on immunogenicity istested. Different human antibody isotypes have different abilities tobind Fc receptors on cells, activate complement, and generally induceeffector functions such as antibody-dependent cellular cytotoxicity andcomplement-dependent cytotoxicity. Antibodies with an isotype that ismore proficient at engaging Fc receptors can be more likely to becaptured by Fc receptors, internalized, proteolytically processed, andpresented as peptides on major histocompatibility complex molecules,thus increasing the probability of a cell mediated immune response thatcan lead to immunogenicity. The effects of isotype on immunogenicity canbe tested in a transgenic animal, such as the XenoMouse® strain of miceexpressing fully human antibodies of several isotypes, by comparingantibodies in which the antibody heavy chain variable region and lightchain are identical, and the antibody heavy chain constant region isvaried to represent different human isotypes (IgG1, IgG2, IgG3, IgG4,IgA, IgE, and/or IgM). In some embodiments, the human immunoglobulintransgenic animals are further genetically modified to express one ormore human Fc receptors. In some embodiments, the relativeimmunogenicity of antibodies differing only in their isotype iscompared.

In some embodiments, the impact of post-translational modifications isexamined. The expression system used to produce antibodies for humantreatment can impact immunogenicity. For example, the selected systemcan result in the addition of non-human glycans to the antibody, or theabsence of glycosylation. Mammalian antibodies possess a complexbiantennary oligosaccharide on heavy chain residue Asn297 (reviewed byChadd and Chamow in Curr. Opin. Biotechnol. 12, 188-194 (2001)).Expression of human antibodies in nonhuman systems—most commonly hamsterCHO cells and mouse NS0 cells and hybridomas—results in distinctglycosylation patterns (id.). Certain expression systems, such as yeastor transgenic livestock, also exhibit differences in glycosylation. Uponadministration to an animal or human subject, this can lead tocomplement activation or antibody binding to mannose-binding lectin.Enhanced binding to the mannose receptor on dendritic cells may enhanceantibody uptake and processing by these professional antigen presentingcells (Dong et al., J. Immunol. 163, 5427-5434 (1999)), therebyincreasing the potential for triggering immunogenicity. Antibodies madein plants and E. coli lack oligosaccharides altogether. In addition, thenature of antibody glycosylation (or lack thereof) may also alterantibody structure (Krapp et al., J Mol. Biol. 325, 979-989 (2003)),thus creating potentially immunogenic conformers. A transgenic animal,such as the XenoMouse® strain of mice expressing fully human antibodies,can be used to assess the effects on immunogenicity of differentglycosylation patterns using different expression systems, keeping theantibody protein sequence constant. As will be appreciated by one ofskill in the art, the antibodies can be made in the particularexpression system, isolated, and then administered to the transgenicanimal for testing.

In some embodiments, the formulation of an antibody preparationinfluences immunogenicity. For example, the formulation can cause anantibody to assume a different tertiary conformation, or to formaggregates. The effects on immunogenicity can be tested in transgenichuman immunoglobulin-expressing mice, such as XenoMouse® mice, byadministering the same dose via the same route of administration, of anantibody with an identical amino acid sequence, which has beenformulated using any of several different approaches. In this manner,data can be obtained that will provide information on the leastimmunogenic formulation method for a given antibody to be administeredto humans.

In some embodiments, the association of dose quantity, frequency, and/orroute of administration of the antibody with the induction of a HAHAresponse is determined. As discussed herein, there is evidence that theamount of antibody given to a subject can affect the risk ofimmunogenicity. An inverse relationship between dose and immunogenicityrisk has been reported. Also, antibody administered after a longerinterval has been reported to cause more immunogenicity than antibodyadministered repeatedly with shorter intervals. The route of antibodyadministration to the subject can also affect immunogenicity, withsubcutaneous administration generally understood to carry a higher riskof immunogenicity than intramuscular or intravenous administration.These aspects can be tested in transgenic humanimmunoglobulin-expressing mice, such as XenoMouse® mice, by comparingthe immunogenicity of a particular chimeric, human, or humanizedantibody in animals that receive the antibody by one of several routes,including but not limited to subcutaneous, intravenous, intraperitoneal,intracranial, intradermal, intramuscular, or oral.

In some embodiments, the immunocompetence of the subject beingadministered the antibody is examined with the transgenic animals. Asappreciated by one of skill in the art, it is understood that anantibody that is either inherently immunogenic, or is administered in away that can cause an immune response to the antibody, may not be seenas immunogenic when administered to a subject whose immune system isimpaired. Consequently, an antibody that has a high incidence ofimmunogenicity in a patient population considered to be immunesufficient, such as humans suffering from chronic inflammatory diseases,can have a low incidence of immunogenicity in a patient population thatis immunosuppressed, such as the elderly or those receiving radio- orchemotherapy regimens. The immunogenicity of an antibody can be relatedto the immune status of the recipient patient. This can be tested intransgenic human immunoglobulin-expressing mice, such as XenoMouse®mice, by comparing the effect of a particular chimeric, human, orhumanized antibody in animals that have been subjected to sublethalirradiation or received chemotherapeutic agents to those that have notbeen manipulated.

In some embodiments, identical antibody proteins are administered to afirst animal that has been pretreated with an antibody that differs fromthe test antibody only in its heavy and light chain variable regions.The first animal also has circulating antibodies that bind to thepre-treatment antibody. The immune response to the subsequent antibodyin the first animal can be compared to the immune response in an animalthat has not been pre-treated in this manner. Thus, pre-treatment of asubject with various antibodies can be one condition that can also beexamined.

Therapeutic Administration and Formulations

A prolonged duration of action will allow for less frequent and moreconvenient dosing schedules by alternate parenteral routes such asintravenous, subcutaneous or intramuscular injection.

When used for in vivo administration, antibody formulations describedherein should be sterile. This is readily accomplished, for example, byfiltration through sterile filtration membranes, prior to or followinglyophilization and reconstitution. Antibodies ordinarily will be storedin lyophilized form or in solution. Therapeutic antibody compositionsgenerally are placed into a container having a sterile access port, forexample, an intravenous solution bag or vial having an adapter thatallows retrieval of the formulation, such as a stopper pierceable by ahypodermic injection needle.

The route of antibody administration is in accord with known methods,e.g., injection or infusion by intravenous, intraperitoneal,intracerebral, intramuscular, intraocular, intraarterial, intrathecal,inhalation or intralesional routes, or by sustained release systems asnoted below. Antibodies are preferably administered continuously byinfusion or by bolus injection.

An effective amount of antibody to be employed therapeutically willdepend, for example, upon the therapeutic objectives, the route ofadministration, and the condition of the patient. Accordingly, it ispreferred for the therapist to titer the dosage and modify the route ofadministration as required to obtain the optimal therapeutic effect.Typically, the clinician will administer antibody until a dosage isreached that achieves the desired effect. The progress of this therapyis easily monitored by conventional assays or by the assays describedherein.

Antibodies as described herein can be prepared in a mixture with apharmaceutically acceptable carrier. Therapeutic compositions can beadministered intravenously or through the nose or lung, preferably as aliquid or powder aerosol (lyophilized). Composition can also beadministered parenterally or subcutaneously as desired. Whenadministered systemically, therapeutic compositions should be sterile,pyrogen-free and in a parenterally acceptable solution having due regardfor pH, isotonicity, and stability. These conditions are known to thoseskilled in the art. Briefly, dosage formulations of the compounds of thepresent invention are prepared for storage or administration by mixingthe compound having the desired degree of purity with physiologicallyacceptable carriers, excipients, or stabilizers. Such materials arenon-toxic to the recipients at the dosages and concentrations employed,and include buffers such as TRIS HCl, phosphate, citrate, acetate andother organic acid salts; antioxidants such as ascorbic acid; lowmolecular weight (less than about ten residues) peptides such aspolyarginine, proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidinone;amino acids such as glycine, glutamic acid, aspartic acid, or arginine;monosaccharides, disaccharides, and other carbohydrates includingcellulose or its derivatives, glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;counterions such as sodium and/or nonionic surfactants such as TWEEN,PLURONICS or polyethyleneglycol.

Sterile compositions for injection can be formulated according toconventional pharmaceutical practice as described in Remington'sPharmaceutical Sciences (18^(th) ed, Mack Publishing Company, Easton,Pa., 1990). For example, dissolution or suspension of the activecompound in a vehicle such as water or naturally occurring vegetable oillike sesame, peanut, or cottonseed oil or a synthetic fatty vehicle likeethyl oleate or the like may be desired. Buffers, preservatives,antioxidants and the like can be incorporated according to acceptedpharmaceutical practice. As will be appreciated by one of skill in theart, the risk that any of the above compositions or materials willreduce, prevent, induce, or exacerbate a HAHA response can also beexamined. This can be achieved in the manner outlined above, where anantibody with a known risk or degree of inducing a HAHA response isadministered to a XenoMouse® mouse, together with the substance to betested. Any increase in the HAHA response, compared to a normalizedlevel of HAHA response, can demonstrate that the substance is adding tothe HAHA response. Alternatively, the substance can be tested alone(without the HAHA response inducing antibody), to determine if it canindependently induce a HAHA response. Of course, the various methods ofadministration, and how they relate to the risk of a HAHA response canalso be examined. For example, the amount, frequency, or time (comparedto other relevant events in the day) of administration can all readilybe examined.

Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing thepolypeptide, which matrices are in the form of shaped articles, films,or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (e.g., poly (2-hydroxyethyl-methacrylate) asdescribed by Langer et al., J. Biomed Mater. Res., (1981) 15:167-277 andLanger, Chem. Tech., (1982) 12:98-105, or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers,(1983) 22:547-556), non-degradable ethylene-vinyl acetate (Langer etal., supra), degradable lactic acid-glycolic acid copolymers such as theLUPRON Depot™ (injectable microspheres composed of lactic acid-glycolicacid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyricacid (EP 133,988).

While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated proteinsremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for protein stabilization depending on themechanism involved. For example, if the aggregation mechanism isdiscovered to be intermolecular S—S bond formation through disulfideinterchange, stabilization may be achieved by modifying sulfhydrylresidues, lyophilizing from acidic solutions, controlling moisturecontent, using appropriate additives, and developing specific polymermatrix compositions.

Sustained-released compositions also include preparations of crystals ofthe antibody suspended in suitable formulations capable of maintainingcrystals in suspension. These preparations when injected subcutaneouslyor intraperitoneally can produce a sustain release effect. Othercompositions also include liposomally entrapped antibodies of theinvention. Liposomes containing such antibodies are prepared by methodsknown per se: U.S. Pat. No. DE 3,218,121; Epstein et al., Proc. Natl.Acad. Sci. USA, (1985) 82:3688-3692; Hwang et al., Proc. Natl. Acad.Sci. USA, (1980) 77:4030-4034; EP 52,322; EP 36,676; EP 88,046; EP143,949; 142,641; Japanese patent application 83-118008; U.S. Pat. Nos.4,485,045 and 4,544,545; and EP 102,324.

The dosage of the antibody formulation for a given patient will bedetermined by the attending physician taking into consideration variousfactors known to modify the action of drugs including severity and typeof disease, body weight, sex, diet, time and route of administration,other medications and other relevant clinical factors. Therapeuticallyeffective dosages may be determined by either in vitro or in vivomethods.

An effective amount of the antibody to be employed therapeutically willdepend, for example, upon the therapeutic objectives, the route ofadministration, and the condition of the patient. Accordingly, it ispreferred that the therapist titer the dosage and modify the route ofadministration as required to obtain the optimal therapeutic effect. Atypical daily dosage might range from about 0.001 mg/kg to up to 100mg/kg or more, depending on the factors mentioned above. Typically, theclinician will administer the therapeutic antibody until a dosage isreached that achieves the desired effect. The progress of this therapyis easily monitored by conventional assays or as described herein.

It will be appreciated that administration of therapeutic entities inaccordance with the compositions and methods set forth herein will beadministered with suitable carriers, excipients, and other agents thatare incorporated into formulations to provide improved transfer,delivery, tolerance, and the like. A multitude of appropriateformulations can be found in the formulary known to all pharmaceuticalchemists: Remington's Pharmaceutical Sciences (18^(th) ed, MackPublishing Company, Easton, Pa. (1990)), particularly Chapter 87 byBlock, Lawrence, therein. These formulations include, for example,powders, pastes, ointments, jellies, waxes, oils, lipids, lipid(cationic or anionic) containing vesicles (such as Lipofectin™), DNAconjugates, anhydrous absorption pastes, oil-in-water and water-in-oilemulsions, emulsions carbowax (polyethylene glycols of various molecularweights), semi-solid gels, and semi-solid mixtures containing carbowax.Any of the foregoing mixtures may be appropriate in treatments andtherapies in accordance with the present invention, provided that theactive ingredient in the formulation is not inactivated by theformulation and the formulation is physiologically compatible andtolerable with the route of administration. See also Baldrick P.“Pharmaceutical excipient development: the need for preclinicalguidance.” Regul. Toxicol. Pharmacol. 32(2):210-8 (2000), Wang W.“Lyophilization and development of solid protein pharmaceuticals.” Int.J. Pharm. 203(1-2): 1-60 (2000), Charman W N “Lipids, lipophilic drugs,and oral drug delivery—some emerging concepts.” J Pharm Sci.89(8):967-78 (2000), Powell et al. “Compendium of excipients forparenteral formulations” PDA J Pharm Sci Technol. 52:238-311 (1998) andthe citations therein for additional information related toformulations, excipients and carriers well known to pharmaceuticalchemists. The amount and route of administration can depend upon theresults obtained from experiments described herein.

EXAMPLES Example 1

This example demonstrates how a normalized host V_(H) gene profile canbe generated. First, one determines which V_(H) genes are present in allof the members of the profile. As the sequence of all the V genes arecurrently known, as well as methods for their identification, this isroutine for one of skill in the art. Next, the frequency of occurrencefor each gene is determined by determining the number of people in whichthe gene occurred and dividing that number by the number of people inthe profile. As shown in FIG. 1A, the genes of five hosts aredetermined, and then normalized in various ways, as shown FIG. 1C. Thisis repeated for each gene to develop a full profile of V genes for agiven population.

Example 2

This example demonstrates how a normalized host V protein profile can begenerated. The normalized host V gene profile of Example 1 is convertedto various amino acid sequences for the genes that are actuallyexpressed. An example of the proteins expressed from the V genes in FIG.1A is shown in FIG. 1B and the resulting frequencies are shown in FIG.1D.

As can be seen in comparing FIG. 1C and FIG. 1D, the frequency and thusthe risk associated with the gene, can vary depending upon whether theanalysis is performed at the DNA level or the protein level.

Example 3

This example demonstrates changes that can be made in order to reducethe risk associated with a selection of genes. Once a high-risk gene isidentified in a selection of genes, as shown in FIG. 3, it can beoptimized. In order to increase the likelihood of reducing a HAHAresponse, and decrease the risk of a loss in functionality, themodification can be directed by information in the normalized host Vprotein profile. An example of this is demonstrated in FIG. 6. In FIG.6, the gene to be optimized is gene C as it has the lowest frequency ofoccurrence (20%). In this example, gene C will be removed. If areplacement gene is required, the replacement of gene C will bedetermined by examining the profile to determine other genes withsimilar protein structures. Here, this can be gene B, or another genethat is more similar to gene B than it is to gene C.

Example 4

This example demonstrates how one can determine the risk associated witha particular V gene, when the V gene is given to a patient in the formof an antibody. First, a normalized host V gene profile is selected forthe population to which the patient belongs. This can be done bydetermining which V genes a patient has or likely has. Next, the V geneof the antibody to be administered to the patient will be determined.Next, the V gene of the antibody will be compared to the V genes in theprofile.

For example, there can be five genes in the profile of the population,A, B, C, D, and E, with frequencies of, gene A at 30%, gene B at 2%,gene C at 5% gene D at 100%, and gene E at 99%. If part of the antibodyis encoded by an A gene, the antibody will be considered a 70% riskgene. If part of the antibody is encoded by a B gene, it will beconsidered a 98% risk gene. If part of the antibody is encoded by a Cgene, it can be considered a 95% risk gene. If part of the antibody isencoded by a D or an E gene, it can be considered a 0 or 1% risk gene.

Example 5

This example describes how one can verify the correlation betweenfrequency of occurrence of a V_(H) gene and the risk that the gene willinduce a HAHA response in a patient.

First, antibodies with known gene sets are administered to patients.Next, the patients are tested for a HAHA response. Antibodies thatinduce a HAHA response will have their gene sets given a point value.All of the genes of all of the antibodies administered will have each oftheir point values totaled. These values will then be normalized to howfrequently the gene occurred. Thus, a gene that will be in 10antibodies, but only one of which caused a HAHA response, will beassigned 0.1 point value, while a gene that is in one antibody, butcauses a HAHA response in all individuals in a given profile will beassigned 1.0 point value. Genes with the highest point value will bethose genes with the greatest association with the induction of a HAHAresponse. These point values will then be compared to the gene frequencyof occurrence a normalized host V gene profile to determine how closethe correlation is. This will demonstrate that common genes have a lowerfrequency of being associated with a HAHA response, and rare genes, in apopulation, have a higher frequency of being associated with a HAHAresponse.

Example 6

The presence of the V_(H)3-9 gene was examined in various samples. Astandard PCR-ELISA technique was used to search for the appearance ofV_(H)3-9 in genomic DNA of a human donor and four human cell lines,A375, A549, MCF7, and HEK-293 cells. The biotin-labeled probe which wasused in PCR-ELISA had the sequence (5′→3′)[BioTEG]CCGGCAAGCTCCAGGGAAGGGC (SEQ ID NO: 14, FIG. 23B). The results,shown in FIG. 10, demonstrate that V_(H)3-9, and variability in thepresence of V_(H)3-9, can be detected through PCR-ELISA with this probe.

Example 7

This example demonstrates that the XenoMouse® animal is useful indetermining if an antibody will induce a HAHA response. Groups ofXenoMouse® mice capable of producing fully human antibodies wereimmunized with a selected fully human antibody. Each mouse receivedthree doses of one of two different antibodies (A or B), spaced twoweeks apart, 10 micrograms antibody per dose. Additionally, some micereceived 10 micrograms keyhole limpet hemocyanin (KLH; Pierce, RockfordIll.) as a positive control immunogen, while others were injected onlywith the carrier as a negative control. Antibody A was an antibodycreated from a XenoMouse® mouse and directed against TNFalpha. AntibodyA was previously disclosed in U.S. Pat. Application No. 60/430,729 (SEQID NO.: 71-74; currently SEQ ID NOs.: 5-8, with SEQ ID NOs.: 12 and 13showing the consensus sequences of the light chain and heavy chainrespectively, FIG. 23B), and identified as 299v2. Antibody B was anantibody that was also directed to TNFalpha. Antibody B is also known asHUMIRA (D2E7, adalimumab), and the antibody was the subject of U.S. Pat.No. 6,258,562, issued to Salfeld et al., Jul. 10, 2001, both hereinincorporated in their entireties by reference. The U.S. Pat. No.6,258,562 characterizes this antibody as a human antibody that binds tohuman TNF alpha. (See generally, the U.S. Pat. No. 6,258,562). However,previous studies have demonstrated that antibody B can elicit a HAHAresponse when administered to humans. (information available on the FDAwebsite, pdf ada1abb123102r1p2.pdf)

The antibody, KLH, or carrier was administered intravenously (“I.V.”),subcutaneously (“S.C.”), or emulsified in complete Freund's adjuvant(Sigma, St. Louis, Mo.) and then administered in the base of the tail(first dose) followed by article emulsified in incomplete Freund'sadjuvant (Sigma) and administered intraperitoneally (second and thirddoses) (“BIP/ADJ”). Serum was obtained from the mice by retro-orbitalbleeds taken at the times indicated in FIGS. 11A-C and FIGS. 12A-C, andstored at −80° C. until all samples could be tested, as described in thefollowing paragraphs.

The serum samples were assayed for the presence of components reactiveto the injected antibody using a bridging ELISA. In this ELISA assay,Maxisorp 96-well plates (Nunc, Rochester, N.Y.) were coated with thetest antibody or KLH, as appropriate, and the plates were then washedand blocked with bovine albumin. XenoMouse® animals' serum samples werethen added in duplicate in a 1:10 dilution. As negative and positivecontrols, 10% normal mouse serum (Equitech-Bio, Kerrville, Tex.) andrabbit anti-human IgG (Southern Biotechnology, Birmingham, Ala.) orXenoMouse® animal-derived anti-KLH (Abgenix) diluted in 10% mouse serum,respectively, were used. Following incubation, the serum was washed awayand a biotinylated form of the test antibody or KLH (prepared using theEZ-Link™ Sulfo-NHS-LC-Biotinylation kit and accompanying protocol,Pierce) was added to the wells, followed by additional washing andincubation with streptavidin coupled to horseradish peroxidase.Visualization was with Enhanced K-blue TMB substrate (Neogen, Lexington,Ky.), and the reaction was stopped with 2 molar sulfuric acid. Plateswere read in a plate reader at 450 nm wavelength. Data are presented asO.D. multiple, which was calculated using the following formula:

$\frac{{Sample}\mspace{14mu}{average}\mspace{14mu}{OD}\mspace{14mu}( {{duplicate}\mspace{14mu}{wells}} )}{{Negative}\mspace{14mu}{control}\mspace{14mu}{average}\mspace{14mu}{OD}\mspace{14mu}( {6 - {8\mspace{14mu}{wells}\mspace{14mu}{per}\mspace{14mu}{plate}}} )}$

O.D. multiples >2 were considered positive for serum antibodies to thetest antibody. Data from two fully human IgG1 test antibodies is shownin FIGS. 11A-11C and FIGS. 12A-12C. BIP/ADJ results are shown in FIGS.11A and 12A, S.C. results are shown in FIGS. 11B and 12B, and I.V.results are shown in FIGS. 11C and 12C.

As can be seen from the data in FIGS. 11A-C, antibody A resulted in noimmunogenicity being detected in the XenoMouse® animals, regardless ofthe method of administration of the antibody. On the other hand, as canbe seen in FIGS. 12A-C, antibody B elicited a high level ofimmunogenicity. The negative control results from the 10% normal mouseserum and the positive control rabbit anti-human IgG were as follows:for Antibody A, negative control had an average OD of 0.05, standarddeviation of 0.01 and OD multiple 1.00; the positive control had anaverage OD of 0.19, standard deviation of 0.02 and an OD multiple of3.81; for Antibody B, negative control had an average OD of 0.07,standard deviation of 0.00, and an OD multiple of 1.00; and the positivecontrol had an average OD of 0.11, standard deviation of 0.02 and an ODmultiple of 1.55. FIG. 12D shows the results from the positive controlimmunogen, KLH, experiment.

As can be seen, while I.V. administration of antibody B elicited animmunogenic response in three of the seven mice, the number of miceexhibiting immunogenicity to antibody B increased to five of seven inthe XenoMouse® animals that received antibody S.C., and finally to sevenof seven for antibody administered with an adjuvant, as shown in FIG.12A. Thus, this example demonstrates that the XenoMouse® animals can beused to distinguish the degree of immunogenicity of two human orhumanized antibodies, both of which are directed to the same protein.

This also demonstrates that antibodies produced from the XenoMouse®mouse will not induce a HAHA response in a host with the same geneprofile. This also demonstrates that various methods of administrationof the antibody to a subject can impact the resulting likelihood that acompound will induce a HAHA response. Additionally, this demonstratesthat the materials added to the subject with the antibody can also alterthe risk or likelihood that an antibody (or the substance itself) willinduce a HAHA response in the subject.

As appreciated by one of skill in the art, many of the above variablesmay be adjusted without altering the general concept taught. Forexample, an O.D. multiple of >2 need not be the precise cutoff. Forexample, the cutoff can be set at 3 standard deviations above thebackground, or more complex statistical methods can be used.

Example 8

This example demonstrates one method for avoiding possible falsenegatives in using a XenoMouse® mouse described herein. A humanizedIgG1,κ antibody, Xolair (E25, Omalizumab) (heavy chain V region, SEQ IDNO.: 10 and light chain region, SEQ ID NO.: 11) was coupled to asynthetic peptide having the sequence CQYIKANSKFIGITELKK (SEQ ID NO.: 9)(herein referred to as “TCE”). Maleimide chemistry was performed usingsulfo-SMCC to generate TCE-decorated antibody (“Ab-TCE”). Additionalantibody was treated identically, but without addition of TCE to thereaction (“Ab-sham”).

Groups of XenoMouse® mice capable of producing fully human antibodieswere immunized with 10 micrograms of either the unmodified, untreatedtest antibody (“Ab”), Ab-TCE or Ab-sham. Additional groups of micereceived 10 micrograms of TCE peptide only as a control. Each mouse wasadministered three doses, spaced two weeks apart, either intravenously(“I.V.”) or subcutaneously (“S.C.”). In addition, unmodified Ab wasemulsified in complete Freund's adjuvant (Sigma, St. Louis, Mo.) andadministered to one group of mice in the base of the tail (first dose)followed by Ab emulsified in incomplete Freund's adjuvant (Sigma)(second and third doses) (“BIP/ADJ”) administered intraperitoneally.Serum was obtained from the mice by retro-orbital bleeds taken at thetimes indicated in the figure below and stored at −80° C. until allsamples could be tested.

The serum samples were assayed for the presence of components, whichincludes antibodies, reactive to the injected antibody using a bridgingELISA. In this ELISA assay, Maxisorp 96-well plates (Nunc, Rochester,N.Y.) were coated with the unmodified test antibody, and the plates werethen washed and blocked with bovine albumin. XenoMouse® animals' serumsamples were then added in duplicate in a 1:10 dilution. As negative andpositive controls, 10% normal mouse serum (Equitech-Bio, Kerrville,Tex.) and rabbit anti-human IgG (Southern Biotechnology, Birmingham,Ala.) diluted in 10% mouse serum were used. Following incubation, theserum was washed away and a biotinylated form of the test antibody(prepared using the EZ-Link™ Sulfo-NHS-LC-Biotinylation kit andaccompanying protocol, Pierce) was added to the wells, followed byadditional washing and incubation with streptavidin coupled tohorseradish peroxidase. Visualization was with Enhanced K-blue TMBsubstrate (Neogen, Lexington, Ky.), and the reaction was stopped with 2molar sulfuric acid. Plates were read in a plate reader at 450 nmwavelength. Data are presented as O.D. multiple, which was calculatedusing the following formula:

$\frac{{Sample}\mspace{14mu}{average}\mspace{14mu}{OD}\mspace{11mu}( {{duplicate}\mspace{14mu}{wells}} )}{{Negative}\mspace{14mu}{control}\mspace{14mu}{average}\mspace{14mu}{OD}\mspace{14mu}( {6 - {8\mspace{14mu}{wells}\mspace{14mu}{per}\mspace{14mu}{plate}}} )}$

O.D. multiples >2 were considered positive for serum antibodies to thetest antibody.

FIGS. 13A-D, FIGS. 14A-D, and FIG. 15 show the results from theexperiment. The results indicate that TCE addition to an antibodyresulted in immunogenicity in 2 of 7 mice, whereas unmodified andsham-conjugated antibody were not immunogenic in 14 mice tested. Thus,TCE conjugation to a protein will allow the reporting of B cell-mediatedimmunogenicity. XenoMouse® mice did produce antibody in response tounmodified antibody when it was administered with adjuvant, suggestingthat the test antibody contains endogenous T cell epitopes.

Example 9

This example demonstrates a normalized host V_(H) gene profile oranalysis of the V_(H) gene repertoire across a population, on bothgenomic DNA and RNA levels, from blood peripheral cells from 96 donors,using gene-specific probes. The presence of transcripts and genomicsequence in PBMC was determined V_(H) gene segments

Probes for the V_(H) genes were designed and validated on plasmidsand/or cDNA from hybridomas expressing the target gene and relatedfamily members.

Donors, PBMC Isolation and Nucleic Acid Preparation

Peripheral blood was obtained from 66 donors and from 32 individualsthrough a commercial source (Bioreclamation, Inc.). Data on age, genderand ethnicity of all donors was collected. Peripheral blood mononuclearcells (PBMC), from 45 ml of blood were isolated. Cells were counted andgenomic DNA was isolated from 5×10e6 cells using Qiagen's DNeasy 96Tissue Kit, according to the manufacturer's instructions. Quantitationwas performed with PicoGreen (from Molecular Probes) and 50 ng of gDNAwas used in real-time PCR reactions. RNA was isolated using RNeasy minicolumns, according to manufacturer's instructions. Residual DNA wasremoved with TURBO DNA-free from Ambion. RiboGreen (Molecular Probes)was used for quantitation of RNA samples and cDNA was made withInvitrogen's SuperScript First-Strand Synthesis System for RT-PCR, using80 ng of total RNA. One-twelfth of the final reaction mixture was usedin real-time PCR analysis.

Primer and Probe Design

Primers and probes were designed based on sequence information availablein Vbase (and shown in FIG. 17A-FIG. 22J), using Primer Express softwareversion 2.0 (Applied Biosystems). Minor Groove Binding (MGB) probes hada Tm of 65-67° C. Where possible, probes were designed in FR1, otherwisethey were in the FR2, FR3 or leader regions. CDR regions and reportedpolymorphisms were avoided. This can be done in a variety of ways, forexample, by sequence comparisons, as shown in FIG. 16A and FIG. 16Bbetween various V_(H) genes. As will be appreciated by one of skill inthe art, any wrap around in FIGS. 16A and 16B (e.g., genes 2-05, 2-26,3-43, and 7.41) is a result of spatial constraints and not meant toindicate alignment properties. PCR amplicons were kept as short aspossible, not to exceed 200 bp. Sequences of the probes and 20 basesupstream and downstream were searched against the human genome databaseaccording to the method referenced in Altschul, Stephen F., Thomas L.Madden, Alejandro A. Schäffer, Jinghui Zhang, Zheng Zhang, Webb Miller,and David J. Lipman (1997), “Gapped BLAST and PSI-BLAST: a newgeneration of protein database search programs”, Nucleic Acids Res.25:3389-3402 to minimize cross-reactivity with pseudogenes.

Real-Time PCR

Real-time PCR was carried out on an ABI Prism 7700 analyzer.Annealing/extension temperature (from 60 to 68° C.) for eachprimer/probe pair was optimized using plasmids or hybridoma cDNA samplescontaining the sequence of interest, and closely related family members.cDNA was analyzed first and if not all donors expressed the gene, gDNAwas analyzed.

The results for V_(H)3-9, V_(H)3-13 and V_(H)3-64 genes are summarizedin Table 1. High risk genes were those that were absent, not expressedin a subpopulation of subjects (e.g., less than 100%), or that had mRNAlevels at lower than detectable levels.

TABLE 1 V gene cDNA gDNA 3-9 90%  91% 3-13 99% 100% 3-64 97% 100%

Based on this data, it can be seen that, for this population, V_(H)3-9occurs with much less frequency that the other two genes. Additionally,that 3-13 and 3-64, while occurring at the genomic level in the entirepopulation, did not appear at the mRNA level for the entire population.

Risk-assessment at an early stage of development can be a valuable toolin the evaluation of potential therapeutic antibodies. The resultspresented here can be used as additional criteria for selection of leadcandidate molecules for (pre)clinical development.

Of course, as will be appreciated by one of skill in the art and asdiscussed in detail above, the groupings into which the various V genesfall can vary depending upon the selected cutoff lines for high-risk andlow-risk genes in the population. For example, with a simple analysis,any time a V gene is less than 100% present as gDNA, it can be a highrisk gene. More preferably, anytime a V gene is less than 100% presentas mRNA, it can be a high risk gene. However, for example, in a morequantitative analysis, anytime a gene is present as cDNA in less than80% of the population, it is a high risk gene. Alternatively, it can bereferred to based on its frequency; thus, instead of high medium or low,a gene that appears in 80% of the population could be a 20% risk gene(or have a 20% probability of inducing a HAHA response in any individualin the population), as the odds that a person in the population will nothave the same gene is 20%. In some embodiments, the values for high andlow risk for populations can vary based on the particular uses of theantibody. However, one of skill in the art will readily be able todecide their desired values based on their particular circumstances andthe teachings herein.

Example 10

The germline V_(k) locus contains 132 genes, with 45 of these possessingan open reading frame, 25 of which are present in XenoMouse® animals.This includes 7 duplicated gene pairs with identical sequences, whichcan be treated as a single gene. Thus, a total of 18 differentfunctional V_(k) gene sequences are present in XenoMouse® animals. Thesame techniques and analysis as shown in Example 9 can be performed onV_(kappa) or V_(lambda) genes. The process will be the same, only thegenes and primers, etc., should be switched. Some of the various V genesthat can be tested are displayed in FIG. 8 and sequences of particular Vgenes, for V_(lambda), and V_(kappa) are shown in FIGS. 19A-19G,20A-20L, 21A-21F, and 22A-22J. The methods are the same as that shown inExample 10 above. The results will reveal which genes are common in thepopulation or individual and which genes are not.

Example 11

This example demonstrates how the XenoMouse® animal can be used todetect other variables that influence the likelihood that a HAHAresponse will occur. The effect of isotype on immunogenicity is testedin a transgenic animal, such as the XenoMouse® strain of mice expressingfully human antibodies of several isotypes. This can be done bycomparing the HAHA response induced by foreign antibodies in which theantibody heavy chain variable region and light chain are identical, andthe antibody heavy chain constant region is varied to representdifferent human isotypes (e.g., IgG1, IgG2, IgG3, IgG4, IgA, IgE). Thevarious antibodies (e.g., first and second foreign antibodies) thatrepresent the various isotypes are each administered to the transgenicanimal and the presence and degree of a HAHA response is measured. Theisotype that demonstrates the greater HAHA response will be the one withthe larger risk of inducing a HAHA response in humans. Of course,alternatively, this could be used to determine the isotype with thelowest risk of inducing a HAHA response.

Example 12

This example demonstrates how the XenoMouse® animal can be used todetect other variables that influence the likelihood that a HAHAresponse will occur. The effect of the expression system onimmunogenicity is tested in a transgenic animal, such as a XenoMouse®strain of mice expressing fully human antibodies. The XenoMouse® strainof mice can be used to assess the effects on immunogenicity of differentglycosylation patterns using different expression systems, keeping theantibody protein sequence constant.

A first foreign antibody can be administered to a first transgenicanimal. The first antibody will be created from an E. coli expressionsystem. Next, a second foreign antibody can be administered to a secondtransgenic animal (that is the same in terms of species and strain). Thesecond foreign antibody will be produced in yeast or transgeniclivestock. A third foreign antibody, created in Chinese hamster ovarycells, can be administered to animals of the same transgenic strain. Theantibody from the expression system that demonstrates the greatest HAHAresponse will be the one with the larger risk of inducing a HAHAresponse.

Example 13

This example demonstrates how the XenoMouse® animal can be used todetect other variables that influence the likelihood that a HAHAresponse will occur.

The effect of the formulation on immunogenicity is tested in transgenichuman immunoglobulin-expressing mice, such as XenoMouse® mice, byadministering the same dose via the same route of administration, of aforeign antibody with an identical amino acid sequence, which has beenformulated using a variety of different approaches (e.g., a firstformulation condition and a second formulation condition). In thismanner, data is obtained that will provide information on the leastimmunogenic formulation method for a given antibody to be administeredto humans.

Example 14

This example demonstrates how the XenoMouse® animal can be used todetect other variables that influence the likelihood that a HAHAresponse will occur.

The impact of the route of administration and the amount of antibodyadministered can also be examined. These can be tested in transgenichuman immunoglobulin-expressing mice, such as XenoMouse® mice, bycomparing the immunogenicity of a particular antibody in animals thatreceive the antibody by one of several routes, including but not limitedto subcutaneous, intravenous, intraperitoneal, intracranial,intradermal, intramuscular, and/or oral. This comparison willdemonstrate the risk associated with the route or amount of antibodyadministered with the risk of a HAHA response. This can also be repeatedwith various amounts of the antibody to determine if certain levels ofadministration are more likely to induce a HAHA response.

As will be appreciated by one of skill in the art, a similar protocol tothat described above can be used where the time interval betweenmultiple doses is altered to determine if certain dosing schedules aremore likely to induce a HAHA response.

Example 15

This example demonstrates how the XenoMouse® animal can be used todetect other variables that influence the likelihood that a HAHAresponse will occur.

How the immunocompetence of the subject being administered the antibodyinfluences the induction of a HAHA response can be examined. This can betested in transgenic human immunoglobulin-expressing mice, such asXenoMouse® mice, by comparing the effect of a particular chimeric,human, or humanized antibody in animals that have been subjected tosublethal irradiation or received chemotherapeutic agents to thoseanimals that have not been so manipulated. First, a first XenoMouse®mouse has its immunocompetence impaired by administering a sublethalamount of radiation to the mouse. Next, a test antibody is administeredto the mouse and any HAHA response from the mouse observed. Next, asecond, unmanipulated and therefore immunocompetent XenoMouse® mouse isadministered the same antibody and any HAHA response from the secondmouse is observed. This comparison will demonstrate the impact ofimmunocompetence on the risk of inducing a HAHA response.

As can be seen from the data herein, there are several genes that can bedescribed as high-risk genes in this particular population. Thissuggests that this approach can be used for various types ofimmunoglobulin genes, for example V_(lambda) genes, as well as D and Jgenes.

INCORPORATION BY REFERENCE

All references cited herein, including patents, patent applications,papers, text books, and the like, and the references cited therein, tothe extent that they are not already, are hereby incorporated herein byreference in their entirety.

EQUIVALENTS

The foregoing description and Examples detail certain preferredembodiments of the invention and describes the best mode contemplated bythe inventors. It will be appreciated, however, that no matter howdetailed the foregoing may appear in text, the invention may bepracticed in many ways and the invention should be construed inaccordance with the appended claims and any equivalents thereof.

1. A transgenic mouse comprising: a genome comprising humanimmunoglobulin gene loci that following their B cell immunoglobulin generearrangements produce at least one human antibody, wherein the humanimmunoglobulin gene loci comprises genes that encode for a humanantibody; and a foreign human antibody, wherein the foreign humanantibody was encoded by a human immunoglobulin gene that is not presentin the genome of the transgenic mouse wherein the foreign human antibodyis capable of inducing a human-anti-human antibody (HAHA) response, andwherein the human antibody produced by the transgenic mouse binds to theforeign human antibody.
 2. The transgenic mouse of claim 1, where thetransgenic mouse has its mouse immunoglobulin genes inactivated.
 3. Thetransgenic mouse of claim 1, wherein the genome lacks at least one humanimmunoglobulin gene, selected from the group consisting of V_(H)3-9,V_(H)3-13 and V_(H)3-64.
 4. The transgenic mouse of claim 3, whereinsaid foreign human antibody is encoded by a gene selected from the groupconsisting of: V_(H)3-9, V_(H)3-13, and V_(H)3-64.
 5. The transgenicmouse of claim 3, wherein the mouse lacks a V_(H)3-9 gene.
 6. Thetransgenic mouse of claim 5, wherein the foreign human antibody isencoded by the VH3-9 gene.
 7. The transgenic mouse of claim 1, furthercomprising a candidate HAHA inhibitor that is inside of said transgenicmouse.
 8. The transgenic mouse of claim 1, wherein the transgenic mousehas its mouse immunoglobulin genes inactivated.
 9. The transgenic mouseof claim 1, further comprising an antigenic substance, wherein theantigenic substance is attached to the foreign human antibody.
 10. Atransgenic mouse comprising: a genome comprising human immunoglobulingene loci that following their B cell immunoglobulin gene rearrangementsproduce at least one human antibody, wherein the human immunoglobulingene loci comprises genes that encode for a human antibody; and aforeign human antibody, wherein the foreign human antibody was encodedby a human immunoglobulin gene that is not present in the genome of thetransgenic mouse, wherein the foreign human antibody is capable ofinducing a human-anti-human antibody (HAHA) response, and wherein thehuman antibody produced by the transgenic mouse binds to the foreignhuman antibody, and wherein at least a portion of the foreign humanantibody is encoded by the human immunoglobulin gene that is notexpressed in the genome of the transgenic mouse.
 11. The transgenicmouse of claim 10, where the transgenic mouse has its mouseimmunoglobulin genes inactivated.
 12. The transgenic mouse of claim 10,wherein the genome lacks at least one human immunoglobulin gene,selected from the group consisting of V_(H)3-9, V_(H)3-13 and V_(H)3-64.13. The transgenic mouse of claim 12, wherein said foreign humanantibody is encoded by a gene selected from the group consisting of:V_(H)3-9, V_(H)3-13, and V_(H)3-64.
 14. The transgenic mouse of claim12, wherein the mouse lacks a V_(H)3-9 gene.
 15. The transgenic mouse ofclaim 14, wherein the foreign human antibody is encoded by the VH3-9gene.